TW202020035A - Highly purified and/or modified fucan compositions for the treatment of fibrous adhesions - Google Patents

Abstract

Compositions, methods, systems, etc., are provided for modified and/or purified fucans and corresponding fucan-containing compositions that inhibit fibrous adhesions among other advantages. The purified/modified fucans and fucan compositions have a reduced level of non- fucan components or impurities such as those found in a starting fucan composition. Such reduced undesirable components or impurities include, for example, undesired components bound to the fucan and compounds in the composition that are not a part of or bound to the fucan.

Description

Highly purified and/or modified polytrehalose composition for the treatment of fibrous adhesions

The present invention relates to highly purified and/or modified polytrehalose compositions for the treatment of fibrous adhesions. Priority claim

This application claims the same U.S. Provisional Patent Application No. 62,711,364 filed on July 27, 2018; U.S. Provisional Patent Application No. 62,711,372 filed on July 27, 2018; July 27, 2018 US Provisional Patent Application No. 62/711,335; US Provisional Patent Application No. 62/713,399 filed on August 1, 2018; US Provisional Patent Application No. 62/722,135 filed on August 23, 2018; U.S. Provisional Patent Application No. 62/755,311 filed on November 2, 2018; U.S. Provisional Patent Application No. 62/793,514 filed on January 17, 2019; U.S. Provisional Patent Application filed on June 13, 2019 No. 62/861,223; U.S. Provisional Patent Application No. 62/713,392 filed on August 1, 2018; U.S. Provisional Patent Application No. 62/713,413 filed on August 1, 2018; U.S. Provisional Patent Application No. 62/722,137 filed on August 23, 2018; U.S. Provisional Patent Application No. 62/755,318 filed on November 2, 2018; U.S. Provisional Patent Application filed on June 13, 2019 No. 62/861,228; U.S. Provisional Patent Application No. 62/755,328 filed on November 2, 2018; U.S. Provisional Patent Application No. 62/793,654 filed on January 17, 2019 and The rights and interests of US Provisional Patent Application No. 62/861,235 filed on June 13, 2019, all of the above applications are incorporated by reference in their entirety.

Polytrehalose (including fucoidan) is a sulfated polysaccharide. Generally speaking, this means that it is a molecule composed of a large number of carbohydrate groups and also has a sulfur atom attached to the carbohydrate group. The main sugar group is called "fucose", which is a sugar with 6 carbon atoms and the chemical formula C ₆ H ₁₂ O ₅ . "Fuccan gum" (or fucoidan) refers to polytrehalose derived from brown algae (algae). Polytrehalose may be present alone or in a mixture of other sugars, for example in the form of a mixture of sugars such as xylose, galactose, glucose:glucuronic acid and/or mannose. These other sugars can be extracted from seaweed or other sources with polytrehalose. Although polytrehalose is currently derived from natural sources, such as the brown algae (seaweed), sea cucumbers, etc. mentioned herein, there is no final source of polytrehalose. "Polytrehalose" includes those with polytrehalose as discussed herein Polymer molecules of chemical and structural elements.

Fucoidan can be obtained from various species of brown algae, including (but not limited to): Adenocystis utricularis , Ascophyllum nodosum , Chorda filum , Cystoseirabies marina , sea velvet ( Durvillaea antarctica ), Echlonia kurome , Ecklonia maxima , Eisenia bicyclis , Fucus evanescens , Fucus vesiculosis , Hizikia fusiforme , Himanthalia Elongata , Kjellmaniella crassifolia , Laminaria brasiliensis , Laminaria cichorioides , Laminaria hyperborea , Laminaria japonica , sugar Laminaria saccharina , Lessonia trabeculata , Macrocystis pyrifera , Pelvetia fastigiata , Pelvetia Canaliculata , Saccharina japonica , broad-leaved sugar Algae ( Saccharina latissima ), Sargassum stenophylum , Sargassum thunbergii , Sargassum confusum , Sargassum fusiforme and Undaria pinnatifida . These exemplary species are all from the taxonomic class Phaeophyceae and most of these species belong to the following families: Fucales and Laminariaceae .

Polytrehalose, including fucoidan, has been shown to effectively serve as a barrier device for preventing, inhibiting and treating fibrous adhesion formation. It has also been found to be used to treat other related diseases and conditions. Therefore, the demand for the preparation of purified polytrehalose is not met, including that the polytrehalose is modified to have the desired molecular weight distribution and/or sulfate content. The compositions, systems and methods of the present invention provide these and/or other advantages.

The present invention provides compositions, methods, systems, and the like for polytrehalose and polytrehalose-containing compositions for inhibiting fibrous adhesion, among other advantages. The poly-trehalose and poly-trehalose compositions herein include poly-trehalose having a specific content of a desired specified poly-trehalose component, including purified/modified poly-trehalose. The subject of this application often refers to purified/modified polytrehalose and purified/modified polytrehalose compositions; except in the scope of patent applications or unless the context clearly restricts to purified/modified polytrehalose/ In addition to the purified/modified polytrehalose composition, this reference includes all polytrehalose/polytrehalose compositions herein. In some embodiments, the polytrehalose and the composition containing polytrehalose are suitable for medical and surgical applications. The poly-trehalose and poly-trehalose compositions have a reduced amount of non-poly-trehalose components or impurities, such as those found in the raw poly-trehalose composition. Such undesirable components or impurities include (for example) undesired components bound to polytrehalose (eg, ionic, covalent, hydrogen bonding, etc.) and parts that are not polytrehalose or are not chemically and/or Or a compound ionically bound to the composition of polytrehalose. Undesired polytrehalose components can be quantified compared to (eg, w/w) polytrehalose. The non-polytrehalose compound and the undesired poly-trehalose component may be collectively referred to as poly-trehalose and/or impurities including the composition of poly-trehalose herein. These purified/modified polytrehalose can reduce dangerous complications due to impurities, for example during the medical and surgical use of polytrehalose.

The compositions, methods, systems, etc. of the present invention provide a polytrehalose composition containing the desired polytrehalose (including the initial or initial polytrehalose composition (ie, from which purified/modified polytrehalose can be obtained The starting polytrehalose composition may or may not be a crude product or has been previously processed, such as the raw material polytrehalose composition) purified/modified polytrehalose) composition and obtaining the required purified /Modified polytrehalose method and method using this purified/modified polytrehalose.

In some aspects, the system, device and method of the present invention provide a composition comprising polytrehalose, which comprises greater than 90% w/w, 92% w/w, 94% w/w, 95% w/w, 97% w/w, 97.5% w/w, 98%, 98.8%, 99%, 99.5% or 99.9% w/w total fucose, galactose, sulfate and relative ion content. In some embodiments, the fucose content may be greater than 25% w/w, 30% w/w, 35% w/w; the galactose content may be less than 10% w/w or 5% w/w. The total relative ion content can be less than 17% w/w or 14% w/w. The relative ion may be a pharmaceutically acceptable relative ion, such as aluminum, arginine, benzathine, chloroprocaine, choline, sodium, potassium, lithium, ammonium, ethylenediamine , Diethylamine, diethanolamine, ethanolamine, histidine, lysine, N-methyl reduced glucosamine, meglumine, procaine, triethylamine, zinc, calcium and magnesium. In some embodiments, the relative ions may include, consist of, or consist essentially of at least one of sodium and potassium, or consist essentially of at least one of sodium and potassium.

In certain embodiments, the provided composition comprising purified/modified polytrehalose may comprise greater than 75% w/w, 80% w/w, 84% w/w total fucose, galactose and Sulfate content. At least 60% w/w may have a molecular weight distribution greater than 100 kDa, a weight average molecular weight greater than 100 kDa and/or a peak molecular weight greater than 70 kDa. An exemplary method for determining this distribution is to use an aqueous gel permeation chromatography setup, which contains or consists essentially of: A 300 mm analytical gel permeation chromatography column with an inner diameter of 7.8 mm, filled with a hydroxylated polymethacrylate based gel, with an effective molecular weight range between about 50 kDa and about 5,000 kDa; one 300 mm analytical gel permeation chromatography column with an inner diameter of 7.8 mm, filled with hydroxylated polymethacrylate-based gel, with an effective molecular weight range between 1 kDa and about 6,000 kDa; and one with A 40 mm protective column with an inner diameter of 6 mm is filled with a gel based on hydroxylated polymethacrylate. The two analytical gel permeation chromatography columns and one protective column are included at approximately 30°C. In the column chamber; Refractive index detector at about 30℃; 0.1M sodium nitrate mobile phase at 0.6 mL/min; and Relative to the peak molecular weight standard curve, the peak molecular weight standard curve basically consists of the following: the first polydextrose standard with a peak molecular weight of about 2,200 kDa; the second polydextrose standard with a peak molecular weight between about 720 kDa and about 760 kDa Between; the third polydextrose standard, the peak molecular weight is between about 470 kDa and about 510 kDa; the fourth polydextrose standard, the peak molecular weight is between about 370 kDa and about 410 kDa; the fifth polydextrose standard, the peak molecular weight is about 180 kDa and about 220 kDa; and the sixth polydextrose standard, the peak molecular weight is between about 40 kDa and 55 kDa. The peak molecular weight standard curve may further include polydextrose standards with peak molecular weights between about 3 kDa and about 5 kDa.

The provided composition comprising polytrehalose may have a number average molecular weight greater than 50 kDa, between 20% w/w and 60% w/w or between 30% w/w and 55% w/w or 35 Sulphation levels between% w/w and 52% w/w.

The total carbohydrate content can be between 27% w/w and 80% w/w. The percentage of total fucose content to total carbohydrate content may be at least about 30% w/w, 50% w/w, 70% w/w, 90% w/w, or 95% w/w. The percentage of total galactose content to total carbohydrate content may be less than about 60% w/w or 20% w/w. The total glucuronic acid, mannose, rhamnose and xylose content as a percentage of the total carbohydrate content may be less than about 30% w/w.

Also provided herein are methods comprising making or using the provided compositions comprising the purified/modified polytrehalose herein. The use may include treatment of fibrous adhesions.

In some embodiments, the polytrehalose composition herein includes a solid polytrehalose composition and comprises at least 90% w/w polytrehalose, such as the purified/modified polytrehalose herein, the solid poly The trehalose composition further comprises less than 7% w/w, 6% w/w, 5% w/w, 4% w/w, 3% w/w, 2% w/w, 1% w/w or 0.1 % w/w total water content.

In certain embodiments, the composition herein is a medically acceptable polytrehalose composition comprising a therapeutically effective amount of the purified/processed herein in a medically acceptable buffer or diluent Modify polytrehalose. The method herein includes the treatment of fibrous adhesions in animals (such as humans). The treatment includes the selection of purified/modified polytrehalose herein to inhibit fibrous adhesions and administration of 0.5 mg/kg to the wound site of the animal. A therapeutically effective amount of the composition or polytrehalose contained in the composition within a dose range between 50 mg/kg of the composition herein. The dose range can also be between 1 mg/kg and 25 mg/kg. The method may also include using a dosage range comprised between 0.5 mg/kg and 50 mg/kg or between 1 mg/kg and 25 mg/kg to treat the target disease or condition, including, for example, fibrous adhesions.

Also provided herein are methods of making compositions comprising the purified/modified polytrehalose herein, such methods including, for example, for use in starting polytrehalose compositions (such as raw polytrehalose compositions) Method for removing impurities to obtain purified/modified polytrehalose. Such methods may include, for example: Provide the starting polytrehalose composition containing impurities; Add flocculation aid to the starting polytrehalose composition to produce a reaction mixture; Flocculate impurities by heating the reaction mixture to produce flocculated impurities; and Remove flocculated impurities.

Providing the starting polytrehalose composition may include providing the starting polytrehalose composition in a solution, and the method may further include collecting the purified/modified polytrehalose in a solution that reduces impurities. Flocculating the impurities may include heating the reaction mixture above atmospheric pressure, and the flocculation aid may include salts, such as alkali metal, alkaline earth metal, aluminum and/or ammonium chloride, bromide, iodide, fluoride, sulfate, subsulfate Sulfate, carbonate, bicarbonate, phosphate, nitrate, nitrite, acetate, citrate, silicate and/or cyanide. The flocculation aid may include alkali, such as hydroxides and/or oxides of alkali metals, alkaline earth metals, aluminum and/or ammonium.

The removed impurities include at least one of the following: granules, lipids, fatty acids, brown algae polyphenols (phlorotannin), laminin, alginates, proteins, Maillard reaction products, fucoxanthin ( fucoxanthin), chlorophyll, bacteria, cellular components and DNA.

Additional methods for removing impurities from the starting polytrehalose composition to obtain purified/modified polytrehalose may include: Provide a solid starting polytrehalose composition and an insoluble polytrehalose extraction medium that is configured to dissolve impurities; Mixing the starting polytrehalose composition with the extraction medium to produce a purified/modified polytrehalose and extraction medium mixture; and The purified/modified polytrehalose is separated from the extraction medium.

The method may further include collecting the composition comprising purified/modified polytrehalose in a solid state. The extraction medium may contain at least one organic solvent having a relative polarity of less than 0.765. The relative polarity value can be normalized according to the measurement of the solvent displacement of the absorption spectrum. See, for example, Christian Reichardt, Solvents and Solvent Effects in Organic Chemistry, Wiley-VCH Publishers, 3rd edition, 2003. The organic solvent may include at least one of the following: ethanol, isopropanol, methanol, benzene, diethyl ether, decamethylcyclo-pentasiloxane (decamethylcyclo-pentasiloxane), ethyl acetate, butanol, hexane, heptane Alkane, heptanol, octanol and decanol. The extraction medium may further include at least one of the following: alkali, detergent, and oxidant. Providing the starting polytrehalose composition in solid form may comprise precipitating the starting polytrehalose composition from the solution. The removed impurities may include at least one of the following: particles, lipids, fatty acids, brown algae polyphenols, laminose, alginate, protein, Mena reaction products, fucoxanthin, chlorophyll, bacteria, cellular components and DNA.

Other methods for removing impurities from the starting polytrehalose composition to obtain purified/modified polytrehalose may include: The starting polytrehalose composition containing impurities contained in the solution, including suspended impurities; Using an ionic polyvalent impurity precipitant to precipitate impurities out of solution, which in turn produces a mixture of suspended impurities, precipitated impurities and supernatant solution; and Separate suspended impurities and Shendian impurities from the supernatant solution. The method may also include collecting a supernatant solution comprising the compositions, the compositions comprising purified/modified polytrehalose.

The ionic multivalent impurity precipitating agent may contain salts of divalent or trivalent cations; the salt may be chloride, bromide, iodide, fluoride, sulfate, sulfite, carbonate, bicarbonate, phosphate, Nitrate, nitrite, acetate, citrate, silicate and/or cyanide. The cation may be alkaline earth metal, zinc, aluminum, copper and/or iron. The ionic multivalent impurity precipitant may contain a base of divalent or trivalent cations. The base may be hydroxides and/or oxides of alkaline earth metals, zinc, aluminum, copper and/or iron.

Separating the suspended impurities and the precipitated impurities from the supernatant solution may further include flocculating the suspended impurities and the precipitated impurities by adding a flocculant to the mixture of suspended impurities, precipitated impurities, and supernatant solution. The flocculant may contain at least one of the following: potassium aluminum sulfate; aluminum sulfate aluminum; aluminum ammonium sulfate; calcium chloride; sodium phosphate; aluminum hydroxide; aluminum chloride; ferric chloride; ferric sulfate; Sodium, calcium silicate, calcium phosphate, zinc chloride, calcium carbonate, calcium bicarbonate, potassium sulfate, magnesium phosphate, acrylamide, acrylic acid, aluminum chlorohydate, polyaluminum chloride, tannin ); Formaldehyde; Melamine; N,N-dimethylaminoethyl acrylate methyl chloride (N,N-dimethylaminoethyl acrylate methyl chloride); N,N-dimethylaminoethyl methacrylate (quaternary methyl chloride) ( N,N-dimethylaminoethyl methacrylate methyl chloride quaternary) and polydiallyldimethyl ammonium chloride. The method may further include maintaining a pH between about 7 and 14. Maintaining the pH may include adding alkali. The removed impurities may include at least one of the following: particles, lipids, fatty acids, brown algae polyphenols, laminose, alginate, protein, Mena reaction products, fucoxanthin, chlorophyll, bacteria, cellular components and DNA.

The method for removing impurities from the starting polytrehalose composition to obtain purified/modified polytrehalose may include: Provide the starting polytrehalose composition containing impurities; Adjust the pH of the starting polytrehalose composition to between about 8 and 14; Adding a cell disrupting agent configured for lysing cell components to the starting polytrehalose composition to produce a reaction mixture comprising the cell disrupting agent, biomolecule lysate and the starting polytrehalose composition; and The cell disrupting agent and biomolecule lysate are removed from the reaction mixture.

Providing the starting polytrehalose composition may comprise providing the starting polytrehalose composition in solution, and the method may further comprise collecting or collecting the solid/composed polytrehalose-containing composition in solid form In a solution to reduce impurities. The cell disrupting agent may comprise a cleaning agent, which may be an anionic cleaning agent, a cationic cleaning agent or a non-ionic cleaning agent. The cleaner may contain at least one of the following: sodium dodecyl sulfate (SDS), benzalkonium chloride, Triton X100®, Triton X114®, Brij® cleaner, Tween® cleaner, sodium deoxycholate And alkylbenzene sulfonate. Removing the cell disrupting agent and biomolecule lysate may include adding a flocculant to the reaction mixture, the flocculating agent configured to flocculate the cell disrupting agent and the biomolecule lysate. Removing the cell disrupting agent may include adding a precipitating agent to the reaction mixture, the precipitating agent being configured to render the cell disrupting agent insoluble in the reaction mixture, thereby producing a precipitate. Removing the biomolecule lysate may include adding a precipitant to the reaction mixture, the precipitant being configured to render the biomolecule lysate insoluble in the reaction mixture, thereby producing a precipitate. The method may further include adding a flocculant to the reaction mixture, the flocculant being configured to flocculate the precipitate. The flocculant may contain at least one of the following: potassium aluminum sulfate; aluminum sulfate aluminum; aluminum ammonium sulfate; calcium chloride; sodium phosphate; aluminum hydroxide; aluminum chloride; ferric chloride; ferric sulfate; Sodium, calcium silicate, calcium phosphate, zinc chloride, calcium carbonate, calcium bicarbonate, potassium sulfate, magnesium phosphate, acrylamide, acrylic acid, aluminum chloride hydrate, polyaluminum chloride, tannin, formaldehyde, melamine, acrylic acid N,N-dimethylaminoethyl methyl chloride; N,N-dimethylaminoethyl methacrylate quaternary methyl chloride and polydiallyldimethyl-ammonium chloride.

Removal of anionic cleaners can include anionic adsorption; removal of cationic cleaners can include cationic adsorption; removal of non-ionic cleaners can include microcell phase separation; and removal of cleaners can include hydrophobic adsorption. Removal of cleaning agents may include: Dilute the reaction mixture until the concentration of the cleaning agent can be lower than the predetermined concentration; and The reaction mixture containing the cleaning agent is subjected to diafiltration through a tangential flow filter filter having a molecular weight cutoff higher than the maximum molecular weight of the cleaning agent.

The method may also include adding a chelating agent to the reaction mixture after providing the starting polytrehalose composition and before removing the cell disrupting agent. The chelating agent may include ethylenediaminetetraacetic acid (EDTA), 2,3-dimercapto-1-propanol, ethylenediamine, porphine, and/or citric acid, and the method may further include removing cells The oxidizer stopper is added to the reaction mixture before the disrupting agent to stop the oxidant in the reaction mixture, or the bacteriostatic agent is added to the reaction mixture after the initial polytrehalose composition is provided and before the cell disrupting agent is removed. The bacteriostatic agent may include sodium sulfite, ethylenediaminetetraacetic acid (EDTA), benzalkonium chloride, ethanol, and/or thiourea. The removed impurities may include at least one of the following: particles, lipids, fatty acids, brown algae polyphenols, laminose, alginate, protein, Mena reaction products, fucoxanthin, chlorophyll, bacteria, cellular components and DNA.

The method for removing impurities from the starting polytrehalose composition to obtain a composition comprising purified/modified polytrehalose may also include: The starting polytrehalose composition containing impurities provided in the aqueous starting solution; Mixing the aqueous starting solution with an organic solvent to produce an aqueous-organic phase mixture; and The aqueous phase-organic phase mixture is separated to obtain an aqueous portion and an organic portion.

The method may further include collecting the aqueous portion comprising purified/modified polytrehalose. The organic solvent may include at least one organic solvent having a relative polarity of less than 0.765, which may be at least one of the following: ethanol, isopropanol, methanol, benzene, decamethylcyclopentasiloxane, ethyl acetate, hexane , Heptanol, octanol, decanol, heptane, isobutyl acetate, anisole, isopropyl acetate, 1-butanol, butyl acetate, methyl isobutyl ketone, pentane, 1-pentanol , Ether and propyl acetate. The removed impurities may include at least one of the following: particles, lipids, fatty acids, brown algae polyphenols, laminose, alginate, protein, Mena reaction products, fucoxanthin, chlorophyll, bacteria, cellular components and DNA.

Methods for modifying the cation content of the starting polytrehalose composition may include: The starting polytrehalose composition provided in the starting solution; and The starting solution is diafiltered through the tangential flow filter filter using the solution of the chelating agent across the tangential flow filter filter to produce a trapped polytrehalose composition.

The chelating agent may include at least one of the following: ethylenediamine-tetraacetic acid (EDTA), 2,3-dimercapto-1-propanol, ethylenediamine, porphine, or citric acid. The retained polytrehalose composition may contain a cationic content consisting essentially of sodium and/or potassium.

The method for removing impurities from the starting polytrehalose composition to obtain purified/modified polytrehalose may further include: Provide the starting polytrehalose composition containing impurities; Subject the starting polytrehalose composition to a suitable pressure higher than 70 bar and a suitable temperature higher than 30°C in a supercritical extractor; and Filling the supercritical extractor with supercritical fluid to remove impurities into the supercritical fluid; and After a predetermined amount of time, the supercritical fluid containing extracted impurities is removed.

The method may further include collecting the purified/modified polytrehalose remaining in the supercritical extractor; the pressure may be between about 70 bar and about 2000 bar, and the temperature may be between about 30 °C and about 300 °C. The starting polytrehalose composition may be liquid or solid. The supercritical fluid may include at least one of the following: carbon dioxide, ethanol, ethane, hydrochloric acid, hydrofluoric acid, sulfuric acid, and nitric acid. The predetermined amount of time may be between about 5 minutes and 50 hours. The removed impurities may include at least one of the following: particles, lipids, fatty acids, brown algae polyphenols, laminose, alginate, protein, Mena reaction products, fucoxanthin, chlorophyll, bacteria, cellular components and DNA.

These and other aspects, features, and implementations are set forth in this application, including the following detailed description and drawings. Unless explicitly stated otherwise, all embodiments, aspects, features, etc., can be mixed and matched, combined, and arranged in any desired manner.

These and other aspects, features, and implementations are set forth in this application, including the following detailed description and drawings. Unless explicitly stated otherwise, all embodiments, aspects, features, etc., can be mixed and matched, combined, and arranged in any desired manner.

The current compositions, systems, methods, etc. presented herein include purified/modified polytrehalose. The composition of the present invention can be effective for medical treatment, postoperative treatment, and disease suppression. In some embodiments, the polytrehalose is fucoidan. The purified/modified polytrehalose of the present invention may itself be a medical device, medical material, combination product or may be included on or in a medical device, medical material, combination product or included in a pharmaceutically acceptable, therapeutic and/or Or in medically effective compositions.

The following paragraphs turn to a brief discussion of some of the compositions contained in this document that contain purified/modified polytrehalose, including those that can be formed by various methods using the starting polytrehalose composition using the methods discussed herein Depending on the method selected, any suitable reaction mixture may be used, such as solutions, suspensions, solids, gels, or other modes of performing the various methods. Composition

In certain embodiments, the current compositions, systems, etc. presented herein provide medically acceptable purified/modified polytrehalose compositions (including therapeutically effective amounts of purified/modified polytrehalose compositions) , For the treatment of fibrous adhesions (such as surgical adhesions) and arthritis, psoriasis, or other diseases as needed. Compositions containing purified/modified polytrehalose may contain more than about 75% w/w total fucose, galactose, and sulfate, for example, more than about 80% w/w and more than 84% w/w total Fucose, galactose and sulfate. In some embodiments, the purified/modified polytrehalose may further comprise at least one relative ion up to at least about 5%, 7%, 9%, 10%, or 11% w/w. In some embodiments, the relative ion is a pharmaceutically acceptable relative ion. In some practical embodiments, the counter ion is ionically bound to the sulfate group present on the polytrehalose. Pharmaceutically acceptable relative ions can include at least one of the following: aluminum, arginine, benzathine, chloroprocaine, choline, sodium, potassium, lithium, ammonium, ethylenediamine, diethyl Amine, diethanolamine, ethanolamine, histidine, lysine, N-methyl reduced glucosamine, meglumine, procaine, triethylamine, zinc, calcium and magnesium. The sulfur-containing components of polytrehalose are bonded via C-O-S bonds. The oxygen in such bonds can be considered to be bound to carbon or sulfur first depending on various factors. As used herein, the term "sulfate" refers to two embodiments.

In certain embodiments, the purified/modified polytrehalose herein comprises at least about 85% w/w, 90% w/w, 94% w/w, 97% w/w, or 98% w/w The fucose, galactose, sulfate and relative ions. Exemplary relative ions include up to about 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% w/w calcium, magnesium, potassium, and/or sodium. In some embodiments, the purified/modified polytrehalose contains at least about 25%, 30%, or 35% w/w fucose. In some embodiments, the purified/modified polytrehalose contains less than about 10%, 5%, or 4% w/w galactose. In some embodiments, the purified/modified polytrehalose consists essentially of or consists of such total fucose, galactose, sulfate, and relative ionic components. In some embodiments, except for fucose and galactose, the polytrehalose herein does not substantially or completely have all sugar components. In some embodiments, the polytrehalose herein does not substantially or completely have one or more of the following: glucuronic acid, mannose, rhamnose, xylose, galactose, or glucose; as stated herein As used in the, "substantially does not have" indication (if present) that such sugar components are low enough that their presence is pharmacologically and medically unimportant.

In some other embodiments, the purified/modified polytrehalose herein can be used in multiple applications, including inhibition, prevention, removal, reduction or other treatment of fibrous adhesions and other targets as well as other diseases and/or diseases shape. Treatment includes polytrehalose reducing or preventing the development of a target disease or other pathology, such as reducing or preventing the formation of fibrous adhesions at the target site, which is typically identified by the surgeon or other practitioner as containing or equivalent Selected target sites that are susceptible to fibrous adhesions (or other diseases or conditions), and also includes eliminating existing diseases or other conditions, including (for example) eliminating existing fibrous adhesions. For such suppression, prevention, removal, reduction, or other treatments, polytrehalose is typically provided in the form of a medically acceptable medical device, combination product, or pharmaceutically effective composition Contains additional components, such as binders, adjuvants, excipients, etc., and (if necessary) additional medically active substances, such as contained in the composition but not linked to polytrehalose and/or can be linked The second drug to polytrehalose.

In still other embodiments, the composition comprising the purified/modified polytrehalose herein may be solid, for example, containing a water content of less than about 7% w/w, such as less than about 6%, 5% w/w , 4% w/w, 3% w/w or 2% w/w water content solid composition.

Any desired, suitable measurement system can be used to measure the molecular weight distribution of the purified/modified polytrehalose. Different systems can use different compositions with substantially the same composition or even use the same batch to obtain different readings or results when measuring in different ways. A suitable measurement system is an aqueous gel permeation chromatography setup, which basically consists of: a 300 mm analytical gel permeation chromatography column with an inner diameter of 7.8 mm, filled with hydroxyl-based polymethacrylic acid Ester gel, with an effective molecular weight range between about 50 kDa and about 5,000 kDa; a 300 mm analytical gel permeation chromatography column with an inner diameter of 7.8 mm, packed with hydroxyl-based polymethacrylate Gel, with an effective molecular weight range between about 1 kDa and about 6,000 kDa; and a 40 mm protective column with an inner diameter of 6 mm, filled with a hydroxylated polymethacrylate-based gel, the two An analytical gel permeation chromatography column and a protective column are included in the column chamber at about 30°C; a refractive index detector at about 30°C; 0.1M flowing at 0.6 mL/min Sodium nitrate mobile phase; and quantified relative to a peak molecular weight standard curve consisting essentially of: the first polydextrose standard with a peak molecular weight of about 2,200 kDa; the second polydextrose standard with a peak molecular weight between about 720 kDa and about 760 kDa Between; the third polydextrose standard with a peak molecular weight between about 470 kDa and about 510 kDa; the fourth polydextrose standard with a peak molecular weight between about 370 kDa and about 410 kDa; the fifth polydextrose standard with a peak molecular weight between Between about 180 kDa and about 220 kDa; and the sixth polydextrose standard, the peak molecular weight is between about 40 kDa and 55 kDa. The peak molecular weight standard curve may further include polydextrose standards with peak molecular weights between 3 kDa and 5 kDa.

The purified/modified polytrehalose herein may have a molecular weight distribution, where at least about 25%, 30%, 40%, 50%, 60%, 70%, 75%, 90%, 92%, 97% or The distribution of 98% w/w is greater than 100 kDa. The purified/modified polytrehalose herein may include polytrehalose having a molecular weight distribution, wherein at least about 50%, 60%, 70%, 80%, or 90% w/w of the distribution is greater than 200 kDa. The purified/modified polytrehalose herein may have a molecular weight distribution in which at least about 25%, 30%, 40%, 50%, 60%, 70%, or 75% w/w of the distribution is greater than 500 kDa. The purified/modified polytrehalose herein may have a molecular weight distribution, wherein at least about 5%, 10%, 20%, 30%, or 40% w/w of the distribution is greater than 1600 kDa.

The purified/modified polytrehalose herein may have the following weight average molecular weight: greater than about 100 kDa, for example between about 100 kDa and about 10,000 kDa, between about 200 kDa and about 8,000 kDa, at about 350 kDa Between about 8,000 kDa, between about 450 kDa and about 8,000 kDa, between about 580 kDa and about 8,000 kDa, or between about 800 kDa and about 2,000 kDa. The purified/modified polytrehalose herein may have a peak molecular weight greater than about 70 kDa, such as between about 70 kDa and about 1200 kDa, between about 100 kDa and about 1200 kDa, between about 200 kDa and Between about 1200 kDa, between about 400 kDa and about 1200 kDa, or between about 400 kDa and about 900 kDa.

The purified/modified polytrehalose herein may have the following number average molecular weight: greater than about 50 kDa, between about 50 kDa and about 1,000 kDa, between about 70 kDa and about 1000 kDa, between about 150 kDa and Between about 1000 kDa, between about 250 kDa and about 1000 kDa, or between about 250 kDa and about 700 kDa.

The purified/modified polytrehalose herein may have the following sulfation levels: between about 10% w/w and 70% w/w, between about 20% w/w and 65% w/w, Between about 30% w/w and 60% w/w or between about 40% w/w and 60% w/w.

The purified/modified polytrehalose herein may have the following total fucose: molar ratio of total sulfate: between about 1:0.5 and 1:4, between about 1:0.8 and 1:3.5 , Between about 1:1 and 1:2.5, between about 1:1.2 and 1:2.0, or between about 1:1.5 and 1:3. The purified/modified polytrehalose herein may have the following total fucose plus galactose:total sulfate molar ratio: between about 1:0.5 and 1:4, between about 1:0.8 and 1 :3.5, between about 1:1 and 1:2.5, between about 1:1.2 and 1:2.0 or between about 1:1.5 and 1:3.

The purified/modified polytrehalose herein may have the following total carbohydrate content: between about 27% w/w and 70% w/w, between about 30% w/w and 80% w/w , Between about 40% w/w and 90% w/w or between about 50% w/w and 96% w/w. The fucose content of the purified/modified polytrehalose as a percentage of the total carbohydrates herein may be about 30% w/w and 100% w/w, about 40% w/w and 95% w/w Occasionally between about 50% w/w and 90% w/w. The galactose content of polytrehalose in this article can be 0% w/w and 60% w/w, between about 5% w/w and 30% w/w, or about 8% w/ between w and 10% w/w. The polytrehalose herein may have the following: glucuronic acid content as a percentage of total carbohydrate content is between about 0% w/w and 10% w/w; mannose content as a percentage of total carbohydrate content is about 0 between% w/w and 7% w/w; rhamnose content as a percentage of total carbohydrate content is between 0% w/w and 4% w/w; and xylose content as a percentage of total carbohydrate content It is between 0% w/w and 20% w/w. The polytrehalose herein may have a total glucuronic acid, mannose, rhamnose, glucose, and xylose content of less than about 30% w/w or less than about 12% w/w.

In some embodiments, when dissolved in water at a concentration of 50 mg/mL, the purified/modified polytrehalose herein has the following viscosity: between about 4 cP and about 50 cP, between about 5 cP and about Between 40 cP, between about 10 cP and about 30 cP, about 15 cP, about 20 cP, and about 25 cP. In certain embodiments, when dissolved in water at 1 mg/mL to 100 mg/mL, the purified/modified polytrehalose herein is formed to be transparent and colorless, transparent and light yellow or transparent and light brown One of the solutions.

The purified/modified polytrehalose herein can be in paste, gel, patch, film, spray, liquid, emulsion, cream, solution, suspension, solid, implant, microsphere or other desired forms provide.

The composition presented herein may be a solid consisting essentially of purified/modified polytrehalose. The purified/modified polytrehalose may consist essentially of fucose, galactose, sulfate and relative ions.

The purified/modified polytrehalose herein may be in the form of a solution containing polytrehalose between about 0.01 mg/mL and about 300 mg/mL, for example, between about 0.1 mg/mL and about 100 mg/mL Time, between about 1 mg/mL and about 50 mg/mL and between about 20 mg/mL and about 80 mg/mL. Polytrehalose may consist essentially of fucose, galactose, sulfate and relative ions.

The purified/modified polytrehalose may be in the form of a gel containing polytrehalose between about 100 mg/mL and about 1000 mg/mL, for example between about 100 mg/mL and about 500 mg/mL and Between about 300 mg/mL and about 800 mg/mL. Polytrehalose may consist essentially of fucose, galactose, sulfate and relative ions.

The purified/modified polytrehalose may be in the form of a film containing polytrehalose between about 100 mg/mL and about 1000 mg/mL, for example between about 100 mg/mL and about 500 mg/mL and at Between about 300 mg/mL and about 800 mg/mL. Polytrehalose may consist essentially of fucose, galactose, sulfate and relative ions.

The purified/modified polytrehalose herein can be administered in the form of components comprising the following medical devices, combination products, and/or pharmaceutical compositions: any number of pharmaceutically acceptable excipients, such as gelatin, hydroxy Propylene methylcellulose, lactose, water for USP injection, sodium chloride, sodium phosphate, sodium citrate, sodium ascorbate, phosphate buffer, citrate buffer, phosphate citrate buffer, pluronic , Cellulose, alginate, acrylate, glucuronic acid, polyethylene glycol, polyglucosamine, injectable excipients and Ringer's injection USP.

The purified/modified polytrehalose herein can be in paste, gel, patch, film, spray, liquid, emulsion, cream, solution, suspension, solid, implant, microsphere or other desired forms Cast.

Purified/modified polytrehalose can be administered via intravenous, intra-articular, intralesional, intravaginal, transrectal, intramuscular, intraperitoneal, subcutaneous, topical, intranasal, intraocular, or oral administration. The purified/modified polytrehalose can be delivered directly to the disease site. The purified/modified polytrehalose can be continuously released to the disease site via controlled release of the self-polymerizable dosage form.

The purified/modified polytrehalose herein may be administered as a component of a pharmaceutical composition comprising purified/modified polytrehalose and at least one other drug. The drug may be at least one of the following: paclitaxel (paclitaxel), cranberry (doxorubicin), camptothecin (camptothecin), etoposide (etoposide), mitoxantrone (mitoxantrone), methotrexate ( methotrexate), menadione, plumbagin, juglone, beta-laperchone cyclosporine, sulfasalazine, steroids, rapamycin ( rapamycin), retinoids, docetaxel, colchicine, antisense oligonucleotides and ribonuclease.

In certain embodiments, the purified/modified polytrehalose herein may have an acetyl content of less than about 5% w/w, less than about 2% w/w, and about 0% w/w. In some embodiments, when using 2D ¹ H- ¹³ C heteronuclear multiple quantum coherence (heteronuclear multiple quantum coherence) at 70° C. with solvent signal suppression on a 600 MHz spectrometer equipped with a 5-mm cold probe , In the range of 1030 ppm carbon size, with 8 increments of 256-512 scans per measurement, the purified/modified polytrehalose contained herein contains substantially 0% w/w acetyl content. method

Provide methods, systems, etc. for purifying and/or modifying polytrehalose, for example, from a starting polytrehalose composition containing polytrehalose, such as a raw polytrehalose composition or other polytrehalose-containing composition. As used herein, "impurity" refers to any component of polytrehalose that is not fucose, galactose, sulfate, or relative ions, and refers to any non-polysaccharide present in the composition containing polytrehalose Trehalose component or compound or substance. Impurities can be bound to polytrehalose, such as proteins that are ionically and/or chemically bound to polytrehalose, sugar residues other than fucose and galactose that are part of the polymer structure of polytrehalose, chemically Other sugars bound to polytrehalose and non-polytrehalose impurities that are not bound to polytrehalose but are present in the starting polytrehalose composition, such as the raw polytrehalose composition. Examples of such impurities include (but are not limited to) particles, lipids, fatty acids, brown algae polyphenols, laminose, alginate, proteins, Mena reaction products, fucoxanthin, chlorophyll, bacteria, cellular components and DNA, Several of them contain chromophores and make brown, yellow and green in the starting polytrehalose composition, and several of them can be ionically and/or chemically bound in the starting polytrehalose composition The part of polytrehalose may be the part of polytrehalose in the starting polytrehalose composition. In certain embodiments, the methods and the like herein can be used to prepare purified/modified polytrehalose comprising: at least about 88% w/w, 89% w/w, 90% w/w, 91% w /w, 92% w/w, 93% w/w, 94% w/w, 95% w/w, 96% w/w, 97% w/w, 97.1% w/w, 98% w/w , 98.8% w/w, 99% w/w, 99.5% w/w or 99.9% w/w fucose, galactose, sulfate and relative ions. In some embodiments, the purified/modified polytrehalose comprises at least about 75%, 78%, 80%, 82%, or 84% w/w fucose, galactose, and sulfate. In some embodiments, the purified/modified polytrehalose comprises less than about 0.1%, 0.5%, 1%, 2.9%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11% or 12% w/w impurities. Some of these impurities can cause dangerous complications after medical and/or surgical use of polytrehalose.

In some embodiments, the present invention presents purified/modified polytrehalose with low levels of impurities, which is suitable for medical and surgical applications, such as preventing fibrous adhesions.

The following paragraphs turn to a brief discussion of some of the methods that can be used to produce the purified/modified polytrehalose herein. Physically induced flocculation

The starting polytrehalose composition containing a high content of impurities (such as the raw material polytrehalose composition) undergoes flocculation of impurities, which can be physically induced flocculation. The method may include: providing a starting polytrehalose composition; adding a flocculation aid to the starting polytrehalose composition to produce a reaction mixture; and flocculating impurities in the starting polytrehalose composition by heating the reaction mixture ; Separating the flocculated impurities from the reaction mixture; and collecting the purified/modified polytrehalose after separation.

Flocculating impurities by heating the reaction mixture may include heating the reaction mixture while subjecting the reaction mixture to a pressure above atmospheric pressure. Suitable flocculation aids include (but are not limited to) salts and/or bases, such as alkali metal, alkaline earth metal, aluminum and/or ammonium chloride, bromide, iodide, fluoride, sulfate, sulfite, carbonic acid Salt, bicarbonate, phosphate, nitrate, nitrite, acetate, citrate, silicate, oxide, hydroxide and/or cyanide, such as sodium chloride, sodium sulfate, potassium chloride , Calcium sulfate, sodium phosphate, sodium nitrate, lithium chloride, lithium nitrate, ammonium chloride, sodium carbonate, sodium hydroxide. Separating the flocculated impurities from the reaction mixture may include one or more of centrifugation, filtration, sedimentation, or hydrodynamic flow separation of the reaction mixture.

The method and the like herein may further include desalting the starting polytrehalose composition before adding the flocculation aid. Desalting may include diafiltration of the starting polytrehalose composition in the form of an aqueous solution through a molecular weight cutoff (MWCO) tangential flow filtration (TFF) filter. Diafiltration can include diafiltration of the starting polytrehalose composition by distilled water. The molecular weight cutoff TFF filter may have a molecular weight cutoff that is less than the desired molecular weight separation point or target in the purified/modified polytrehalose or used for the purified/modified polytrehalose, for example 50 kDa, 70 kDa, 100 kDa , 200 kDa, 300 kDa, 500 kDa or 1000 kDa molecular weight cutoff.

The method can be performed in alkaline and neutral environments. Since polytrehalose is easily degraded in an acidic environment, adding a flocculation aid to the starting polytrehalose composition may therefore include making the starting polytrehalose composition alkaline to prevent or inhibit the starting polytrehalose composition The polytrehalose in the material is degraded. In other embodiments, the method can be performed by maintaining the reaction mixture at or near pH 7 or greater.

In some embodiments, the starting polytrehalose composition in solution may be provided. Examples of polytrehalose suitable for processing by the above method include, but are not limited to, fucoidan, and the concentration of polytrehalose in solution may be between 0.01% w/v and 50% w/v. Impurities that can be removed by the above methods include (but are not limited to) particles, lipids, fatty acids, brown algae polyphenols, laminose, alginate, proteins, Mena reaction products, fucoxanthin, chlorophyll, bacteria, cell groups Points and DNA. Solid Phase Extraction

The polytrehalose in the starting polytrehalose composition (such as the raw material polytrehalose composition) containing undesired levels of impurities (including extremely high levels of impurities in the original raw material composition) undergoes solid phase extraction. The method may include: providing a starting polytrehalose composition containing impurities and an insoluble polytrehalose in solid form, an extraction medium configured to dissolve impurities; mixing the starting polytrehalose and the extraction medium to form A mixture of dissolved solid polytrehalose composition and extraction medium containing dissolved impurities; separating purified undissolved solid polytrehalose from the extraction medium containing dissolved impurities; and removing purified from the extraction medium /Modified polytrehalose and collect purified/modified polytrehalose in solid form. Separation may include one or more of the following: for example centrifugation, filtration, sedimentation, and hydrodynamic fluid separation.

The extraction medium may include, for example, at least one of the following: alkali, detergent, and oxidant. Suitable extraction media that do not dissolve polytrehalose include organic solvents with a relative polarity of less than 0.765, such as ethanol, isopropanol, methanol, benzene, diethyl ether, demethylcyclopentasiloxane, ethyl acetate, butanol, hexane Alkane, heptane, heptanol, octanol and decanol. Suitable bases include, but are not limited to sodium hydroxide, potassium hydroxide, lithium hydroxide, and calcium hydroxide. Suitable oxidants include, but are not limited to, one or more of the following: hydrogen peroxide, urea peroxide, and oxidative bleach, including sodium hypochlorite. Suitable cleaners include (but are not limited to) nonionic surfactants, such as Tween®, Brij®, and Triton® series cleaners; anionic surfactants, such as sodium dodecyl sulfate (SDS), deoxycholic acid Sodium; and cationic surfactants, such as benzalkonium chloride (BAC). Specific polytrehalose suitable for the methods herein include, but are not limited to, fucoidan. The mixing of the original (eg starting) polytrehalose composition and the extraction medium can be extended from one minute to 120 hours.

The method may further comprise desalting the starting polytrehalose composition before providing the starting polytrehalose composition in solid form. Desalting may include diafiltration of the starting polytrehalose composition in the form of an aqueous solution through a molecular weight cutoff (MWCO) tangential flow filtration (TFF) filter. Diafiltration can include diafiltration of the starting polytrehalose composition by distilled water. The molecular weight cutoff TFF filter may have a molecular weight cutoff that is less than the desired molecular weight separation point or target in the purified/modified polytrehalose or used for the purified/modified polytrehalose, for example 50 kDa, 70 kDa, 100 kDa , 200 kDa, 300 kDa, 500 kDa or 1000 kDa molecular weight cutoff. Diafiltration may further comprise pre-filtering the starting polytrehalose composition through a suitable pre-filter to remove particulate matter. The method may further comprise lyophilizing a suitable starting polytrehalose composition in solution before providing the starting polytrehalose composition in solid form. The method may further comprise precipitating a suitable starting polytrehalose composition from the solution before providing the starting polytrehalose composition in solid form. Suitable precipitants include (but are not limited to) ethanol, isopropanol, propanol, acetone, methanol, dimethyl sulfoxide, dimethylformamide, ethylene glycol, tetrahydrofuran, acetonitrile, ethylene glycol dimethyl ether, The solubility of diethylene glycol dimethyl ether, dioxane, and polytrehalose decreases as the polarity of the precipitation fluid decreases. Impurities that can be removed by the above methods include (but are not limited to) particles, lipids, fatty acids, brown algae polyphenols, laminose, alginate, proteins, Mena reaction products, fucoxanthin, chlorophyll, bacteria, cell groups Points and DNA. Chemically induced precipitation

Starting polytrehalose compositions (such as raw polytrehalose compositions) containing high levels of impurities (including, for example, suspended particles) undergo chemically induced precipitation of impurities. In certain embodiments, the method may include: providing the starting polytrehalose composition in the starting solution; precipitating the impurities from the starting solution with the help of an ionic polyvalent impurity precipitant to obtain suspended impurities, precipitated impurities Mixture of supernatant and supernatant; separation of suspended impurities and precipitated impurities from the supernatant solution; and after separation of suspended impurities and precipitated impurities from the supernatant, collect the supernatant containing the desired purified/modified polytrehalose液液。 Liquid solution.

Suitable impurity precipitants include ionic polyvalent salts and/or divalent and trivalent cation bases. Examples of such suitable salts include, but are not limited to, alkaline earth metals, zinc, aluminum, copper and iron chlorides, bromides, iodides, fluorides, sulfates, sulfites, carbonates, bicarbonates, Phosphate, nitrate, nitrite, acetate, citrate, silicate and/or cyanide. Examples of such suitable bases include, but are not limited to, hydroxides and/or oxides of alkaline earth metals, zinc, aluminum, copper, and/or iron. Separation of suspended impurities and sediment impurities from the supernatant solution may include flocculation of impurities in the mixture. Suitable flocculants include (but are not limited to) potassium aluminum sulfate; aluminum sulfate; aluminum ammonium sulfate; calcium chloride; sodium phosphate; aluminum hydroxide; aluminum chloride; ferric chloride; ferric sulfate; ferrous sulfate; Sodium, calcium silicate, calcium phosphate, zinc chloride, calcium carbonate, calcium bicarbonate, potassium sulfate, magnesium phosphate, acrylamide, acrylic acid, aluminum chloride hydrate, polyaluminum chloride, tannin, formaldehyde, melamine, acrylic acid N , N-dimethylaminoethyl methyl chloride; N,N-dimethylaminoethyl methacrylate quaternary methyl chloride and polydiallyldimethyl-ammonium chloride. In some embodiments, as can be seen from the foregoing list of flocculants, the flocculant may be an impurity precipitant. Separating precipitated, suspended, and/or flocculated impurities from the supernatant solution may include at least one of the following: centrifugation, filtration, sedimentation, and hydrodynamic flow separation of the mixture of impurities and supernatant solution.

The method may further comprise desalting the starting polytrehalose composition before providing the starting polytrehalose composition. Desalting may include diafiltration of the starting polytrehalose composition in the form of an aqueous solution through a TFF filter. Diafiltration can include diafiltration of the starting polytrehalose composition by distilled water. Diafiltration may include diafiltration of the starting polytrehalose composition through a TFF filter with a MWCO of 5 kDa, 10 kDa, 30 kDa, 50 kDa, 70 kDa or 100 kDa. Diafiltration may further comprise pre-filtering the starting polytrehalose composition through a suitable pre-filter to remove particulate matter.

The method may further comprise maintaining the pH between about 7 and 14 to inhibit or prevent the degradation of polytrehalose in an acidic environment. Maintaining the pH between about 7 and 14 may include adding a suitable base, such as sodium hydroxide. A suitable base can be added to the starting polytrehalose composition, and then the impurities are precipitated from the solution by means of an ionic polyvalent impurity precipitation agent. In other embodiments, after the impurities are precipitated from the solution by means of an ionic polyvalent impurity precipitant, a suitable base may be added to the mixture of precipitated impurities and supernatant solution. In still other embodiments, after separating suspended impurities and sediment impurities from the supernatant solution, a suitable base may be added to the supernatant solution.

Examples of polytrehalose suitable for processing by the above method include, but are not limited to, fucoidan, and the concentration of polytrehalose in solution may be between 0.01% w/v and 50% w/v. Impurities that can be removed by the above methods include (but are not limited to) particles, lipids, fatty acids, brown algae polyphenols, laminose, alginate, proteins, Mena reaction products, fucoxanthin, chlorophyll, bacteria, cell groups Points and DNA. Lysis and flocculation

The starting polytrehalose composition with high content of impurities (such as the raw material polytrehalose composition) undergoes lysis and flocculation. The method in this example may include: providing a starting polytrehalose composition; making the starting polytrehalose composition alkaline; adding a cell disrupting agent to the starting polytrehalose composition to produce a reaction mixture, the cells are disrupted The agent lyses the cell components in the polytrehalose composition and releases them into the lysate of the alkaline reaction mixture containing biomolecular components; the cell disrupting agent and at least a portion of impurities are removed from the reaction mixture to leave Purified polytrehalose required for degradation.

Removal of the cell disrupting agent may include any one or more of the following: precipitation, flocculation, tangential flow filtration, microcell phase separation, ion adsorption, and hydrophobic adsorption. Removal of impurities can include any one or more of the following: precipitation, flocculation, tangential flow filtration, microcell phase separation, ion adsorption, and hydrophobic adsorption. Any of these removal methods or combinations of removal methods may include centrifugation, filtration, sedimentation, or hydrodynamic flow to separate any mixture of solid and liquid phases.

Suitable cell disrupting agents include (but are not limited to) anionic, nonionic and cationic cleaners, such as sodium dodecyl sulfate (SDS), benzalkonium chloride, Triton X100®, Triton X114®, Brij® cleaners, Tween® detergent, sodium deoxycholate and alkylbenzene sulfonate.

In one embodiment of these methods, the cell disrupting agent is sodium dodecyl sulfate (SDS) and removing the cell disrupting agent includes adding a precipitating agent to render the cell disrupting agent insoluble in the alkaline reaction mixture and thereby precipitating the cells Crushing agent. In this embodiment, removing the cell disrupting agent may further include adding the flocculating agent to the reaction mixture to flocculate the precipitated cell disrupting agent and at least a portion of impurities. Removal of the cell disrupting agent may further include centrifugation after flocculation.

Suitable precipitating agents for sodium lauryl sulfate and alkylbenzene sulfonates include, but are not limited to, potassium hydroxide, potassium chloride, calcium chloride, calcium carbonate, and barium chloride. Suitable flocculants include (but are not limited to) potassium aluminum sulfate; aluminum sulfate; aluminum ammonium sulfate; calcium chloride; sodium phosphate; aluminum hydroxide; aluminum chloride; ferric chloride; ferric sulfate; ferrous sulfate; Sodium, calcium silicate, calcium phosphate, zinc chloride, calcium carbonate, calcium bicarbonate, potassium sulfate, magnesium phosphate, acrylamide, acrylic acid, aluminum chloride hydrate, polyaluminum chloride, tannin, formaldehyde, melamine, acrylic acid N , N-dimethylaminoethyl methyl chloride; N,N-dimethylaminoethyl methacrylate quaternary methyl chloride and polydiallyldimethyl-ammonium chloride.

It should be understood here that cell disrupting agents may undergo changes during the Shendian process. For example (but not limited to), if the cell disrupting agent is sodium dodecyl sulfate (SDS), the precipitating agent may be potassium hydroxide (KOH) and as part of the sinking process, the sodium cation may be replaced by potassium, resulting in Potassium dodecyl sulfate is insoluble in the reaction mixture and therefore precipitates. The dodecyl sulfate cation, which functions as the cell disruption part of SDS, remains intact during this process.

In yet other embodiments of the method, the cell disrupting agent may be one or more of sodium dodecyl sulfate (SDS) and sodium deoxycholate and removing the cell disrupting agent includes anion adsorption. Anion adsorption may include adding a suitable positively charged adsorbent for a suitable amount of time, followed by removing the adsorbent. Anion adsorption may further comprise flowing the reaction mixture at a suitable flow rate through a column or filter packed with a suitable positively charged adsorbent.

In yet other embodiments of the method, the cell disrupting agent may be benzalkonium chloride and removing the cell disrupting agent includes cationic adsorption. Cationic adsorption can include adding a suitable negatively charged adsorbent for a suitable amount of time, followed by removing the adsorbent. Cationic adsorption may further comprise flowing the reaction mixture at a suitable flow rate through a column or filter packed with a suitable negatively charged adsorbent.

In yet other embodiments of the method, the cell disrupting agent may be one or more of Triton X100®, Triton X114®, Brij®, and Tween® cleaning agents and removing the cell disrupting agent includes microcell phase separation. Microcellular phase separation may involve changing the temperature of the reaction mixture so that the temperature of the reaction mixture exceeds the cloud point of the cell disrupting agent. Microcell phase separation may include centrifuging the reaction mixture to obtain the desired phase separation.

In other embodiments of the method, the cell disrupting agent may be sodium dodecyl sulfate (SDS), benzalkonium chloride, Triton X100®, Triton X114®, Brij® detergent, Tween® detergent, deoxycholate Any one or more of sodium and alkylbenzene sulfonate, and removing the cell disrupting agent includes one or more of a combination of hydrophobic adsorption and dilution and tangential flow filtration (TFF). Hydrophobic adsorption may include adding a suitable hydrophobic adsorbent for a suitable amount of time, followed by removing the adsorbent. Hydrophobic adsorption may include flowing the reaction mixture at a suitable flow rate through a column or filter packed with a suitable hydrophobic adsorbent. Removal by dilution and TFF can include diluting the reaction mixture so that the cell disrupting agent is reduced below its critical cell concentration, and thus can be adapted by allowing the penetration of the cell disrupting agent from the retentate containing polytrehalose The tangential flow filtration of the molecular weight cutoff (MWCO) TFF filter removes it. Removal by dilution and removal of TFF may involve diafiltration of the reaction mixture through a TFF filter in a suitable amount of diavolume.

The method may further include adding a chelating agent to the reaction mixture to chelate free multivalent cations in the reaction mixture. The chelating agent may be added after providing the starting polytrehalose composition and before removing the cell disrupting agent. The method may further include stopping the oxidant in the reaction mixture. Stopping the oxidant may include adding the oxidant stopper to the reaction mixture before or after removing the cell disrupting agent.

The method may include adding a bacteriostatic agent to the reaction mixture. The bacteriostatic agent can be added after providing the starting polytrehalose composition and before removing the cell disrupting agent. Suitable bacteriostatic agents include, but are not limited to, sodium sulfite, ethylenediaminetetraacetic acid (EDTA), benzalkonium chloride, ethanol, and thiourea.

Suitable chelating agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), 2,3-dimercapto-1-propanol, ethylenediamine, porphin, and citric acid. Suitable oxidizer stoppers include (but are not limited to) sulfite, nitrite, and phosphite. As is evident from the above, several of the listed compounds can have more than one function in these methods.

Suitable hydrophobic adsorbents include, but are not limited to, activated carbon, diatomaceous earth, acrylate nonionic resin, polystyrene nonionic resin, styrene-divinylbenzene (DVB) nonionic resin. Suitable anionic adsorbents include (but are not limited to): amine functional styrene-DVB resin, amine functional methacrylate resin, amine functional methyl methacrylate resin, amine functional butyl methacrylate resin, Amine functionalized agarose resin, amine functionalized polydextrose resin, amine functionalized ceramic resin, amine functionalized silicate and lipid removal agent (LRA).

In some embodiments, the starting polytrehalose composition in solution may be provided. Examples of polytrehalose suitable for processing by the above methods include, but are not limited to, fucoidan. The starting polytrehalose composition may have a polytrehalose concentration in a solution state greater than 0.1% w/v and less than 30% w/v. The cell disrupting agent may have a concentration in a solution state of greater than 0.1% w/v and less than 60% w/v. Impurities that can be removed by the above methods include (but are not limited to) particles, lipids, fatty acids, brown algae polyphenols, laminose, alginate, proteins, Mena reaction products, fucoxanthin, chlorophyll, bacteria, cell groups Points and DNA. Liquid - liquid extraction

The poly-trehalose in the starting poly-trehalose composition (such as the raw material poly-trehalose composition) containing impurities in an undesirable amount is subjected to liquid-liquid extraction. Such methods may include: providing a starting polytrehalose composition in an aqueous starting solution; mixing the starting solution with an organic solvent to obtain an aqueous phase-organic phase mixture, the aqueous phase-organic phase mixture having a purified /The aqueous portion of the modified polytrehalose and the organic portion containing hydrophobic impurities; the aqueous portion is separated from the organic portion; and the aqueous portion containing the purified/modified polytrehalose is collected.

The method may further include desalting the starting polytrehalose composition before mixing the aqueous starting solution with the organic solvent. Desalting may include diafiltration of the starting polytrehalose composition in the form of an aqueous solution through a molecular weight cutoff (MWCO) tangential flow filtration (TFF) filter. Diafiltration can include diafiltration of the starting polytrehalose composition by distilled water. The molecular weight cutoff TFF filter may have a molecular weight cutoff that is less than the desired molecular weight separation point or target in the purified/modified polytrehalose or used for the purified/modified polytrehalose, such as 5 kDa, 10 kDa, 30 kDa , 50 kDa, 70 kDa, 100 kDa, 200 kDa, 300 kDa, 500 kDa or 1000 kDa molecular weight cutoff. Diafiltration may further comprise pre-filtering the starting polytrehalose composition through a suitable pre-filter to remove particulate matter.

Mixing the aqueous starting solution with the organic solvent may include shaking the aqueous organic solvent mixture, stirring the aqueous organic solvent mixture, exposing the aqueous organic solvent mixture to higher shear stress, recycling the aqueous phase into the organic phase, and recycling the organic phase Into the water phase.

Separating the aqueous portion from the organic portion may include at least one of the following: centrifugation, decantation, separatory funnel separation, and hydrodynamic flow separation.

Suitable organic solvents for this method include those with a relative polarity of less than 0.765, such as heptane, isobutyl acetate, anisole, isopropyl acetate, 1-butanol, butyl acetate, methyl isobutyl Ketone, pentane, 1-pentanol, ethyl acetate, ether and propyl acetate. The organic phase may contain impurities such as (but not limited to) lipids, fatty acids, brown algae polyphenols, proteins, fucoxanthin, and/or chlorophyll. Diafiltration

The polytrehalose in the starting polytrehalose composition (such as the raw material polytrehalose composition containing impurities in undesirable levels) undergoes diafiltration. The method may include: subjecting the starting solution containing the starting polytrehalose composition to diafiltration using a chelating agent solution through a first tangential flow filter filter to produce a first retained polytrehalose composition and a chelated cation group Permeated solution; and using the second diafiltration solution to filter the first retained polytrehalose composition through the second tangential flow filtration filter to separate the residual chelating agent from the first retained polytrehalose composition, thereby A second retained polytrehalose composition containing the desired purified/modified polytrehalose is produced. Passing the first entrapped polytrehalose composition through the second tangential flow filter filter may include diafiltration through the first tangential flow filter filter. That is, the same filter can be used in both diafiltration methods.

Subjecting the starting polytrehalose composition to diafiltration may include pre-filtering the starting polytrehalose composition through a pre-filter to remove undesired particulate material. Passing the starting polytrehalose composition through a chelating agent may include subjecting the starting polytrehalose composition to diafiltration by one of the following: ethylenediamine-tetraacetic acid (EDTA), 2,3- Dimercapto-1-propanol, ethylenediamine, porphine or citric acid.

The starting polytrehalose composition may have a polytrehalose concentration in a solution state greater than 0.1% w/v and less than 30% w/v. The chelating agent may have a concentration in a solution state of greater than 0.1% w/v and less than 60% w/v. The resulting first and/or second retention composition may comprise a cation content consisting essentially of sodium and/or potassium.

FIG. 1 shows a schematic diagram of a cationic content modification system 1200 for obtaining a modified cationic content and/or level of the starting polytrehalose composition. The starting polytrehalose composition in a solution state is supplied to the polytrehalose container 1216 through the input supply line 1202 . A suitable solvent containing the starting fucoidan can be pre-filtered through a pre-filter 1204 to remove any undesired particulate matter. The standard size of the pre-filter will typically be larger than the largest polymer molecule to be separated by means of the cation content modification system 1200 .

The TFF input pump 1214 pumps the starting polytrehalose composition to the TFF filter 1210 via the TFF supply line 1212 . The TFF filter 1210 is typically supplied in the form of a cartridge designed to allow input fluid supplied to it to pass through its filter on its retention side, while allowing filtrate to drain through an output line and allowing treated input fluid It exits via another output line as a retentate. The TFF input pump 1214 provides a pressure level on the TFF filter 1210 between its retentate and the permeate side. In FIG. 1, the retentate of the TFF filter 1210 is returned to the polytrehalose container 1216 via the TFF retentate return line 1218 and the TFF retentate valve 1217 , while the filtrate is generated through the TFF filtrate output line 1219 for external cation content Modify system 1200 or discard.

When the TFF input pump 1214 recirculates the pre-filtered fucoidan and retentate through the TFF filter 1210 , the chelating agent from the first diafiltration solution container 1220 can be passed through the first diafiltration solution supply line 1225 , such as (but not Limited to) Ethylenediaminetetraacetic acid (EDTA), 2,3-dimercapto-1-propanol, ethylenediamine, porphine or citric acid is added to the polytrehalose container 1216 containing the starting polytrehalose in. The chelating agent is used for both: to prepare the solvent lost through the filtrate on the TFF filtrate output line 1219 and/or to ensure that a predetermined volume of diafiltration volume of input polytrehalose and chelating agent are circulated through the TFF filter 1210 . As the chelate compound then passes through the TFF filter 1210 into the filtrate, the chelating agent sequesters the cations, specifically multivalent cations, in the starting polytrehalose composition. By controlling the first diafiltration solution valve 1224 , a chelating agent can be added during the pulse. In other embodiments, the chelating agent can be added in a continuous mode. The diafiltration volume of the chelating agent treated by the TFF filter 1210 can be predetermined. The process may last for a predetermined period of time, such as between about 1 and about 6 hours, between about 3 and about 12 hours, and between about 10 and about 24 hours. The process can be continued for a predetermined amount of diafiltration volume of chelating agent, such as between about 1 and about 4 diafiltration volumes, between about 3 and about 6 diafiltration volumes, between about 5 and about 10 diafiltration volumes And between about 7 and about 20 diafiltration volumes. The process is sustainable and can measure the cation content in the polytrehalose container 1216 , and when the desired cation content has been obtained, for example, the cation content contains less than 10 parts per million (ppm), less than 1 ppm, less than Multivalent cations of 0.1 ppm and less than 0.01 ppm terminate the TFF process. The starting polytrehalose composition in solution state was diafiltered through the TFF filter 1210 using the first diafiltration solution to obtain a first retained polytrehalose composition having a modified cation content.

The next step in the process is to remove residual chelating agent from the first retained polytrehalose composition in the polytrehalose container 1216 . This can be accomplished by closing the first diafiltration solution valve 1224 , the cation content modification system output valve 1206, and allowing the second diafiltration solution from the second diafiltration solution container 1230 to pass through the second diafiltration solution supply line 1235 and the second diafiltration The solution valve 1234 enters the polytrehalose container 1216 and is operated. As described previously, the mixture in the polytrehalose container 1216 is then subjected to TFF through the TFF filter 1210 via the TFF supply line 1212 , the TFF input pump 1214 , the TFF retentate return line 1218, and the TFF retentate valve 1217 . The second diafiltration solution may include, for example, but not limited to, any one or more of deionized water, bacteriostatic solution, and salt. The bacteriostatic agent may be, for example (but not limited to) sodium sulfite, EDTA, benzalkonium chloride, ethanol, thiourea. Suitable salts include but are not limited to sodium chloride, potassium chloride, sodium phosphate, ammonium bicarbonate, phosphate buffered saline.

The second diafiltration solution is used for both: preparing the solvent lost through the filtrate on the TFF filtrate output line 1219 and/or the first retained polytrehalose composition and the second diafiltration ensuring a predetermined diafiltration volume The solution is circulated through the TFF filter 1210 . By controlling the second diafiltration solution valve 1234 , the second diafiltration solution can be added during the pulse. In other embodiments, the second diafiltration solution can be added in a continuous mode. The diafiltration volume of the second diafiltration solution processed by the TFF filter 1210 can be predetermined. The process may last for a predetermined period of time, such as between about 1 and about 6 hours, between about 3 and about 12 hours, and between about 10 and about 24 hours. The process can be continued for a predetermined amount of diafiltration volume of chelating agent, such as between about 1 and about 4 diafiltration volumes, between about 3 and about 6 diafiltration volumes, between about 5 and about 10 diafiltration volumes And between about 7 and about 20 diafiltration volumes. For example, the cation content is below 10 parts per million (ppm), below 1 ppm, below 0.1 ppm and below 0.01 ppm multivalent cations. The process is sustainable and the residual chelating agent concentration in the polytrehalose container 1216 can be measured, and suitable lower residual chelating agent concentrations have been obtained, such as below 10 ppm, below 1 ppm, below 0.1 ppm and below At a residual chelating agent content of 0.01 ppm, the TFF process is terminated. The resulting second retained polytrehalose composition in the polytrehalose container 1216 contains the purified/modified polytrehalose product of the process of the cationic content modification system 1200 . If desired, the resulting poly trehalose container 1216. The second retentate trehalose polyethylene composition to modify the system output may be removed via line 1208 from poly cation content of trehalose in the container 1216. Supercritical fluid extraction

The poly-trehalose in the poly-trehalose composition (such as the starting poly-trehalose composition containing undesired levels of impurities) undergoes supercritical fluid extraction. The method may include; providing the starting polytrehalose composition in a solid state; placing the starting polytrehalose composition in a supercritical extractor; and subjecting the starting polytrehalose composition in the supercritical extractor to a high level At a suitable pressure of 70 bar; heating the starting polytrehalose composition in the supercritical extractor to a suitable temperature above 30°C; filling the supercritical extractor with supercritical fluid to produce purified/modified polytrehalose and Supercritical fluid containing extracted impurities; removing the supercritical fluid containing extracted impurities from the supercritical extractor after a predetermined amount of time; and recovering purified/modified polytrehalose.

Filling the supercritical extractor with supercritical fluid may include filling the supercritical extractor with carbon dioxide. Supercritical carbon dioxide can be supplemented with ethanol between 2% v/v and 10% v/v. In some embodiments, the supercritical carbon dioxide may be supplemented with ethanol of about 5% v/v as a co-solvent. Alternative supercritical liquids for carbon dioxide for use with this method include (but are not limited to) ethanol, ethane, hydrochloric acid, hydrofluoric acid, sulfuric acid, and nitric acid.

Subjecting the starting polytrehalose to a suitable pressure may comprise subjecting the starting polytrehalose composition to a pressure between about 70 bar and about 2000 bar. Subjecting the starting polytrehalose composition to a suitable temperature may include subjecting the starting polytrehalose composition to a temperature between about 30°C and about 300°C.

The removal of the supercritical fluid containing extracted impurities after a predetermined amount of time may be included between about 5 minutes and about 50 hours, such as between about 10 minutes and about 1 hour, between about 30 minutes and about 5 hours, The supercritical fluid is removed after about 1 hour to about 24 hours and after about 5 hours to about 48 hours.

The method may further include desalting the starting polytrehalose composition before placing the starting polytrehalose composition in the supercritical extractor. Desalting may include diafiltration of the starting polytrehalose composition in the form of an aqueous solution through a molecular weight cutoff (MWCO) tangential flow filtration (TFF) filter. Diafiltration can include diafiltration of the starting polytrehalose composition by distilled water. The molecular weight cutoff TFF filter may have a molecular weight cutoff that is less than the desired molecular weight separation point or target in the purified/modified polytrehalose or used for the purified/modified polytrehalose, for example 50 kDa, 70 kDa, 100 kDa , 200 kDa, 300 kDa, 500 kDa or 1000 kDa molecular weight cutoff. Diafiltration may further comprise pre-filtering the starting polytrehalose composition through a suitable pre-filter to remove particulate matter. Chemical structural modification

The methods, systems, etc. discussed herein may include chemical structural modifications to polytrehalose, including polytrehalose-containing polytrehalose compositions. Chemical structural modifications may involve the removal of functional groups from polytrehalose, such as the removal of O-acetyl, N-acetyl, methoxy, hydroxyl, carboxylic acid, and/or sulfate radicals from polytrehalose structures Functional group. Chemical structural modifications may involve the use of multiple chemical agents, such as acids, bases, detergents, and/or oxidants. Tangential flow filtration • Some of the methods discussed in this article utilize tangential flow filtration (TFF). Consistent with the typical identification of tangential flow filtration (TFF) filters, the nominal molecular weight cutoff (MWCO) value for a given TFF filter will selectively retain on its cutoff side molecules that contain molecules that have not passed through the filter barrier The solution, and therefore generally has a molecular weight and/or size greater than the molecular weight of the molecules that pass/permeate the barrier to the permeate side. Therefore, the molecular weight cutoff value of a TFF filter is typically not absolute for any given polymer or nominal cutoff value: a given TFF filter will pass or retain some molecules above and below both the nominal molecular weight cutoff. The actual cutoff/selectivity value and impact of the nominal TFF filter for a specific polymer can be routinely determined for the specific polymer. • A large number of factors can affect the penetration behavior of TFF filters. These factors may depend on the TFF filter itself or on the properties of the target polymer, for example, the folding behavior and folding structure of the target polymer may affect the behavior of the target polymer in the MWCO barrier that passes/does not pass through the TFF filter . As I know, with regard to the TFF filter itself, a large number of factors can affect the penetration behavior of the TFF filter. For example, the manufacturing method may result in various pore sizes in a particular TFF filter, and variations thereof may include two pores larger and smaller than the nominal MWCO. Therefore, a TFF filter with a nominal molecular weight cut-off value will essentially pass/retain molecules at the nominal molecular weight cut-off value, but may also pass/retain some molecules below and/or above such values. Gel permeation chromatography • Gel permeation chromatography is used to evaluate the molecular weight distribution obtained in the experimental examples. There are a large number of different parameters, columns, and standards that can be used in gel permeation chromatography, making various instrument settings available for molecular weight analysis. For the molecular weight determination in this article, the following parameters were used for GPC: The mobile phase was 0.1 M sodium nitrate flowing at 0.6 mL/min. The column chamber and detector are at 30°C. Waters 2414 refractive index detector is used for detection. • Suitable GPC columns include GPC columns compatible with aqueous solvents, such as columns packed with at least one of the following: sulfonated styrene-divinylbenzene, NH functionalized acrylate copolymer network, Modified silica and gel based on hydroxylated polymethacrylate. For the analysis in this article, three columns are used in series, which contains a 40 mm long protective column with an inner diameter (ID) of 6 mm, filled with a 6 pm particle size based hydroxylated polymethacrylate gel; It is the first 300 mm analytical GPC column with a 7.8 mm ID, packed with a 12 pm particle size hydroxylated polymethacrylate based gel, which has a discharge limit of about 7,000 kDa and between about 50 kDa and about 5,000 kDa Effective molecular weight range between; followed by a second 300 mm analytical GPC column with a 7.8 mm ID, filled with a 10 pm particle size based hydroxylated polymethacrylate gel, which has a discharge limit of about 7,000 kDa and An effective molecular weight range between about 1 kDa and about 6,000 kDa. The total effective molecular weight range of the column setup is between about 1 kDa and about 6,000 kDa. An example of this string arrangement can be an Ultrahydrogel® protection-Ultrahydrogel® 2000-Ultrahydrogel® linear string connected in series. • Quantify sample flow relative to a standard curve containing traceable standards from American Polymer Standards Corporation: DXT3755K (peak molecular weight = 2164 kDa), DXT820K (peak molecular weight = 745 kDa), DXT760K (peak molecular weight =621 kDa), DXT670K (peak molecular weight=401 kDa), DXT530K (peak molecular weight=490 kDa), DXT500K (peak molecular weight=390 kDa), DXT270K (peak molecular weight=196 kDa), DXT225K (peak molecular weight=213 kDa), DXT150K (peak molecular weight=124 kDa), DXT55K (peak molecular weight=50 kDa), DXT50K (peak molecular weight=44 kDa) and DXT5K (peak molecular weight=4 kDa), the peak molecular weights of these standards are between about 4 kDa and about 2,200 kDa between. The standard curve used may include, for example, at least one of Dextran 3755 kDa, Dextran 50 kDa and Dexran 55 kDa and between 3 and 6 additional traceable standards discussed herein, the calibration point is the peak value of the calibrator used Molecular weight. An example calibration curve may consist of the following: DXT3755K, DXT 820K, DXT530K, DXT500K, DXT225K and DXT55K. The column used herein has a total effective molecular weight range that covers and extends beyond the standard peak molecular weight range for the quantification of polytrehalose. • The molecular weight of the polytrehalose/fucose gum polymer used in this article is the molecular weight value that will always have a higher and lower molecular weight molecular distribution, as the distance between the molecular weight and the specified molecular weight increases or decreases Increase or decrease in number or percentage. The distribution may (but is not required) have the usual Gaussian or distorted Gaussian shape. • The results in the table in this article contain abbreviations for certain characteristics of molecular weight distribution. Gel permeation chromatography is represented by GPC, peak residence time is represented by PRT, peak molecular weight is represented by PMW, weight average molecular weight is represented by WAMW, number average molecular weight is represented by NAMW, distribution percentage is represented by% distribution, molecular weight is represented by MW, and more The dispersibility index is represented by PDI and the molecular weight cutoff is represented by MWCO. Diseases and pathological fibrous adhesions

Fibrous adhesions are a type of scar formed between two parts of the body, usually after surgery (surgical adhesions). Fibrous adhesions can cause serious problems. For example, fibrous adhesions involving female reproductive organs (ovaries, fallopian tubes) can lead to infertility, difficulties with sexual intercourse, and severe pelvic pain. Fibrous adhesions that occur in the intestine can lead to intestinal blockage (obstruction/blockage), and fibrous adhesions can also form in other locations, such as around the heart, spine, and hands. In addition to surgery, fibrous adhesions can be caused, for example, by endometriosis, infection, chemotherapy, radiation, trauma, and cancer.

Various fibrous adhesions are discussed in this document. Such as surgical adhesions, post-surgical adhesions (post-surgical adhesions/postoperative adhesions), adhesions caused by pelvic inflammatory diseases, adhesions caused by mechanical injuries, adhesions caused by radiation, adhesions caused by radiation treatment The terms adhesion caused by trauma and adhesion caused by the presence of foreign materials all refer to the adhesion of tissues caused by similar mechanisms to each other and are included in the term fibrous adhesion.

Fibrous adhesions form a complex process in which normally separated tissues grow into each other. Surgical adhesions (also known as post-operative adhesions) develop from the tissue's otherwise normal wound healing response to trauma and have been reported to occur in more than two-thirds of all abdominal surgery patients (Ellis, H., Surg. Gynecol. Obstet. 133: 497 (1971)). The outcome of these fibrous adhesions is variable and depends on the surgical site or other sites involved, such as the disease site. Problems can include chronic pain, intestinal blockage and even increased risk of death after myocardial surgery (diZerega, GS, Prog. Clin. Biol. Res. 381: 1-18 (1993); diZerega, GS, Fertil. Steril. 61: 219-235 (1994); Dobell, AR, Jain, AK, Ann. Thorac. Surg. 37: 273-278 (1984)). In women of reproductive age, fibrous adhesions involving the uterus, fallopian tubes, or ovaries are estimated to account for approximately 20% of all infertility conditions (Holtz, G., Fertil. Steril. 41: 497-507 (1984); Weibel, MA And Majno, G. Am. J. Surg. 126: 345-353 (1973)).

The formation of fibrous adhesions initially involved the establishment of a fibrin framework and normal tissue repair. The normal repair process allows fibrinolysis along the mesothelial repair. However, in the formation of fibrous adhesions, the fibrin matrix matures as fibroblasts proliferate into a network and angiogenesis occurs, resulting in the establishment of organized fibrous adhesions in about 3 to 5 days (Buckman, RF, et al. , J. Surg. Res. 21: 67-76 (1976); Raferty, AT, J. Anat. 129: 659-664 (1979)). Inflammatory processes include neutrophil activation in traumatic tissues, fibrin deposition and adjacent tissue binding, macrophage invasion, proliferation of fibroblasts into the area, collagen deposition, angiogenesis, and establishment of permanent fibrous adhesion tissue.

Various attempts have been made to prevent surgical adhesions. These involve pharmacological methods aimed at influencing the biochemical and cellular events that accompany surgical trauma and barrier methods for separating affected tissues. For example, the use of peritoneal lavage, heparinized solutions, procoagulants, modification of surgical techniques, such as the use of microscopic or laparoscopic surgical techniques, elimination of talc from surgical gloves, use of smaller sutures, and the use of minimal serous membranes The physical barrier layer (membrane, gel or solution) juxtaposed on the surface has been tried. Currently, preventive therapy also includes preventing fibrin deposition, reducing inflammation (steroidal and nonsteroidal anti-inflammatory drugs), and removing fibrin deposits.

Intervention attempts to prevent the formation of post-operative adhesions have included the use of hydroflotation techniques or barrier devices. Water flotation involves combining a larger volume of polymer solution, such as polydextrose (Adhesion Study Group (Adhesion Study Group), Fertil. Steril. 40:612-619 (1983)) or carboxymethyl cellulose (Elkins, TE, Et al., Fertil. Steril. 41:926928 (1984)) perfused into the surgical space in an attempt to keep the organs apart. Synthetic barrier membrane made of oxidized regenerated cellulose (for example, Interceed™), polytetrafluoroethylene (Gore-tex surgical membrane), and modified glucuronic acid/carboxymethyl cellulose (HA/CMC) combination The fully resorbable film (Seprafilm™) has also been used to reduce post-operative adhesion formation in both animals and humans (Burns, JW, et al., Eur. J. Surg. Suppl. 577: 40-48 (1997); Burns, JW, et al., Fertil. Steril. 66:814-821 (1996)); Becker, JM, et al., J. Am. Coll. Surg. 183:297-306 (1996)). The success of these HA/CMC membranes may stem from their ability to provide tissue separation during the peritoneal wound repair process when fibrous adhesions are formed. It was observed that within 3 to 5 days after application (a time period compatible with the time course of post-operative adhesion formation), the membrane formed a transparent sticky coating on the injured tissue (Ellis, H., Br. J. Surg. 50: 10-16 (1963)). Unfortunately, these methods have had limited success.

Peritonitis involves inflammation of the peritoneum. Peritonitis can cause serious problems. For example, abdominal pain, abdominal tenderness, and abdominal protection. Peritonitis may involve spontaneous, anatomical, and/or peritoneal dialysis-related inflammation. Peritonitis can involve infections such as perforation of hollow organs, peritoneal rupture, spontaneous bacterial peritonitis, and systemic infections can cause infections and peritonitis. Peritonitis may not involve infection, such as the leakage of sterile body fluid into the peritoneum, and sterile abdominal surgery may cause peritonitis. Various attempts have been made to prevent and/or treat peritonitis. For example, general supportive measures such as intravenous rehydration, antibiotics, and surgery. Failure to meet the needs of compounds, compositions, methods, and the like (including delivery methods) that inhibit or otherwise treat and/or prevent (preferably more effectively and with very low side effects) peritonitis.

The purified/modified polytrehalose discussed herein can be used to treat fibrous adhesions in a patient and can be included as a purified/modified polytrehalose medical device, combination or configured and constructed to treat fibrous adhesions Or a component of a pharmaceutical product. For example, it contains between about 0.02 mg/mL and about 100 mg/mL dissolved in physiological saline solution, such as 0.1 mg/mL, 0.2 mg/mL, 0.3 mg/mL, 0.5 mg/mL, 0.9 mg/ mL, 1 mg/mL, 2.5 mg/mL, 5 mg/mL, 7.5 mg/mL of the purified/modified polytrehalose medical device herein. The physiological saline solution may be, for example, lactated Ringer's injection USP (LRS), standard saline, and physiological polydextrose solution.

The purified/modified polytrehalose medical device, which may be a liquid medical device herein, may contain pharmaceutically acceptable excipients, such as buffers, stabilizers, preservatives, adjuvants, and the like. Such purified/modified polytrehalose medical devices can be used by administering polytrehalose from the previous paragraph of about 0.01 mL/kg (according to the patient or target kilogram body weight) to about 10 mL/kg or 15 mL/kg Medical devices to treat fibrous adhesions before, during or after surgery. Doses include, for example, about 0.03 mL/kg, 0.1 mL/kg, 0.2 mL/kg, 0.4 mL/kg, 0.5 mL/kg, 0.6 mL/kg, 1 mL/kg, 1.2 mL/kg, 2 mL/kg, 3 The purified/modified polytrehalose medical devices of mL/kg, 4 mL/kg, 5 mL/kg, 8 mL/kg, 10 mL/kg and 15 mL/kg reach the surgical site of the patient. In other embodiments, such purified/modified polytrehalose medical devices can be used to treat any disease by administering between about 0.04 mg/kg or 0.1 mg/kg to about 25 mg/kg or 50 mg/kg Select target sites, such as fibrous adhesions at lesions, abrasions, injured sites, surgical sites, and post-operative sites. Some examples of such dosages include, for example, about 0.04 mg/kg, 0.075 mg/kg, 0.1 mg/kg, 0.2 mg/kg, 0.5 mg/kg, 1 mg/kg, 1.3 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 7.5 mg/kg, 8 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg and 50 mg/kg The poly-trehalose (including, for example, the purified/modified poly-trehalose herein) reached the surgical site of the patient. The administration can be achieved, for example, by: pouring the liquid medical device into substantially the entire target area; guiding the liquid medical device to a specific location within the target area; spraying the liquid medical device over the entire target area or target area At a specific location within; or via an applicator (which can be a spray applicator through a trocar, catheter, endoscope or other minimally invasive device) spray or otherwise deliver the liquid medical device to the surgeon or Other practitioners have identified specific locations that are particularly susceptible or concerned about fibrous adhesions. In another aspect, the administration can be performed after opening the surgical wound but before the surgical procedure; during the surgical procedure; or after the surgical procedure but before closing the surgical wound. If necessary, the liquid medical device can also be administered after the operation is completed (eg, via a syringe and needle) and can also be administered to a non-surgical target site. The surgical site of the patient may be, for example, at least one of the following: pelvic cavity, abdominal cavity, dorsal vertebral cavity, cranial cavity, spinal cavity, abdominal cavity, thoracic cavity, pleural cavity, pericardial cavity, skin, joints, muscles, tendons, and ligaments. The purified/modified polytrehalose medical device can be administered to the patient's surgical site in less than about 15 minutes, 10 minutes, 8 minutes, 6 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, 1 minute, 45 Seconds, 30 seconds, 20 seconds, 15 seconds, 10 seconds, and 5 seconds.

Examples of administering purified/modified polytrehalose medical devices to the surgical site after opening the surgical wound, during surgery, before closing the surgical wound, and/or after closing the surgical wound include (but are not limited to) the following surgical procedures Purified/modified polytrehalose medical devices are administered at the surgical site: cesarean section surgical procedures, microvascular free flap reconstruction surgery procedures, full-thickness skin graft surgery procedures, VY advancement flap surgery procedures, fascia rotation flap surgery procedures , Arthroplasty surgical procedures, mastectomy surgical procedures, sequestrectomy surgical procedures, saucerization surgical procedures, osteotomy surgical procedures, osteoplasty surgical procedures, Patellectomy surgery procedures, synovectomy surgery procedures, capsulectomy surgery procedures, tendon or ligament repair surgery procedures, tenolysis surgery procedures, tendon amputation surgery procedures , Fasciotomy operation procedure, meniscus repair operation procedure, vertebrectomy operation procedure, ethmoidectomy operation procedure, Caldwell Luc's operation operation procedure, tears Dacryocystorhinostomy surgery procedure, nasal adhesion separation (lysis nasal synechia) surgery procedure, thymectomy (thymectomy) surgery procedure, pneumonolysis (pneumonolysis) surgery procedure, pneumonectomy (pneumonectomy) surgery procedure, Thoracoplasty surgical procedures, bilobectomy surgical procedures, portal hypertension surgery surgical procedures, splenectomy surgical procedures, esophagectomy surgical procedures, peritonitis Surgical procedures, peritonitis surgery procedures, gastrectomy surgery procedures, jejunojejunostomy surgery procedures, laparoscopic cholecystectomy surgery procedures, laparoscopic common bile duct exploration (Laparoscopic common bile duct exploration) Surgical procedures, gastrointestinal anastomosis (gastroentero stomy surgical procedures, bariatric surgery surgical procedures, bowel resection and anastomosis surgical procedures, segmental hepatectomy surgical procedures, lobectomy surgical procedures, pancreatectomy (Pancreatomy) surgical procedure, pancreaticoduodenectomy surgical procedure, tumor resection surgical procedure, laparoscopic nephrectomy surgical procedure, cystectomy surgical procedure, abdomen or pelvis Adhesion separation surgery procedures, hysterosalpingostomy surgery procedures, salpingoplasty surgery procedures, ectopic pregnancy surgery surgery procedures, joint replacement surgery surgery procedures, fracture repair surgery procedures, uterus Hysterectomy surgical procedures, gallbladder removal surgical procedures, heart bypass surgical procedures, angioplasty surgical procedures, atherectomy surgical procedures, breast biopsy surgical procedures, neck Carotid endarterectomy surgical procedures, cataract surgery surgical procedures, coronary artery bypass surgical procedures, dilation and curettage surgical procedures, hernia repair ( hernia repair surgical procedures, lower back pain surgery surgical procedures, partial colectomy surgical procedures, prostatectomy surgical procedures and tonsillectomy surgical procedures. General cancer

Cancer has become the second leading cause of death in the United States and accounts for more than 20% of all deaths. Cancer is a proliferative disease and it is characterized by the uncontrolled division of certain cells, which can lead to the formation of one or more tumors. A large number of methods are used to treat cancer, including surgery, radiation, chemotherapy, and combinations thereof. Although surgery is a relatively common method for some local tumors, there is still a significant chance of tumor recurrence after tumor resection.

Treatment of cancer and other proliferative diseases has been limited by potential damage or toxicity to non-cancerous, healthy tissue. In radiation and surgery, the procedure has been largely restricted to the tumor site and close to the tumor site. However, there may be a significant risk for patients undergoing surgery to remove cancerous tissue (for example, in the removal of prostate or brain tumors, there may be a significant risk of irreparable damage to surrounding important tissues, such as through potential reduction of excision The demand for tumor tissue. In addition, there is a similar risk in concentrated radiation therapy that has been given as a first-line treatment for prostate cancer. In chemotherapeutic treatment of cancer, drugs have been administered systemically, exposing the entire body to drugs. These drugs are designed to be toxic to cancer cells, but they are also toxic to non-cancer cells (generally), making patients very weak when undergoing drug treatment for cancer. Through trials, oncologists can give Some patients can tolerate these doses of drugs. However, these doses usually cannot successfully treat cancer.

One problem with any method of treating cancer is the local recurrence of the disease. For example, approximately 700,000 Americans are diagnosed with local cancer each year (about 64% of all cancer patients) and approximately half a million patients are treated with surgical methods. Unfortunately, 32% of patients treated after surgery relapsed after initial treatment (approximately 21% relapsed at the initial surgical site and 11% relapsed at the distant metastatic site). Nearly 100,000 patients die each year from local recurrence of cancer. This is especially true in breast cancer, where 39% of patients undergoing mastectomy will experience local recurrence of the disease.

Staging is a method to determine the progress of cancer (solid tumor) in a patient. The simplified method divides patients into three groups or stages based on the degree of cancer progression:

Stage 1 : Cancer can be treated by surgically removing some organs. This is also called resectable period.

Stage 2 : The cancer has progressed beyond the resectable point but is still limited to the organ itself.

Stage 3 : The tumor has spread to other organs.

Many cancers are treated with antiproliferative agents, including (for example) 5-fluorouracil (Efudex®), vinca alkaloids (for example, vincristine (Oncovin®)), anthracycline ( anthracyclines) (for example, cranberries (Adriamycin®)), cisplatin (Platinol-AQ®), gemcitabine hydrochloride (Gemzar®), methotrexate, and paclitaxel. Some examples of toxicity associated with anti-proliferative agents, methotrexate, and paclitaxel are discussed elsewhere herein. Methotrexate has been used to treat several types of cancer, including (for example) bladder cancer, breast cancer, cervical cancer, head and neck cancer, liver cancer, lung cancer and testicular cancer. Paclitaxel has been used to treat several types of cancer, including, for example, ovarian cancer, breast cancer, and non-small cell lung cancer (Compendium of Pharmaceutical and Specialties 35th Edition, 2000).

Toxicity caused by 5-fluorouracil can include cardiovascular toxicity, such as myocardial ischemia; central nervous system toxicity, such as euphoria, acute cerebellarsyndrome, and ataxia; dermatology Toxicity, such as alopecia and dermatitis; gastrointestinal toxicity, such as nausea, vomiting, and oral or gastrointestinal ulcers; blood toxicity, such as leukopenia, thrombocytopenia, and anemia; allergic toxicity, such as systemic allergic reactions and contact allergies; eyes Local toxicity, such as increased tearing, photophobia and conjunctivitis; and other toxicity, such as fever. 5-fluorouracil has been used to treat a variety of cancers, including (for example) breast cancer, colorectal cancer, stomach cancer, liver cancer, bladder cancer, head and neck cancer, non-small cell lung cancer, ovarian cancer, pancreatic cancer and prostate cancer ( Compendium of Pharmaceutical and Specialties 35th Edition, 2000).

Toxicity caused by vincristine includes central nervous system toxicity, such as childhood epilepsy and hallucinations; dermatological toxicity, such as alopecia; extravasation toxicity, such as blisters; gastrointestinal toxicity, such as nausea, vomiting, constipation, and stomatitis; Hematological toxicity, such as bone marrow suppression; neurotoxicity, such as peripheral neuropathy and autonomic neuropathy; ocular toxicity, such as diplopia, transient blindness, and eye atrophy; renal/metabolic toxicity, such as urinary retention, hyperuricemia Symptoms and bladder weakness; respiratory toxicity, such as shortness of breath; and other toxicity, such as fever in children. This antiproliferative agent has been used to treat several types of cancer, including (for example) Hodgkin's disease (Hodgkin's disease), small cell lung cancer, Wilm's tumor (Wilm's tumor) and testicular cancer (Compendium of Pharmaceutical and Specialties third 15th edition, 2000).

Toxicity due to cranberries includes cardiovascular toxicity, such as abnormal ECG and cardiomyopathy; dermatological toxicity, such as alopecia and nail changes; extravasation hazard toxicity, such as blisters; gastrointestinal toxicity, such as nausea, vomiting, and oral cavity Inflammation; urogenital toxicity, such as red urine; blood toxicity, such as bone marrow suppression; allergic toxicity, such as systemic allergic reactions and rash; ocular toxicity, such as conjunctivitis; reproductive toxicity, such as infertility; and other toxicity, such as high uric acid Hememia. This antiproliferative agent has been used to treat several types of cancer, including, for example, breast cancer, small cell lung cancer, and ovarian cancer (Compendium of Pharmaceutical and Specialties 35th Edition, 2000).

Toxicity due to cisplatin includes cardiovascular toxicity, such as changes in electrocardiogram; dermatological toxicity, such as hyperpigmentation; extravasation hazard toxicity, such as irritation; gastrointestinal toxicity, such as nausea and vomiting; Anemia; allergic toxicity, such as allergic; myotoxicity, such as peripheral neuropathy and acute encephalopathy; ocular toxicity, such as retrobulbar neuritis; ototoxicity, such as hearing loss and tinnitus; renal/metabolic toxicity, such as toxic Kidney disease and hypokalemia; and other toxicity, such as infertility. This antiproliferative agent has been used to treat several types of cancer, including (for example) bladder cancer, small cell lung cancer, ovarian cancer, testicular cancer, brain cancer, breast cancer, cervical cancer, head and neck cancer, hepatoblastoma cancer and Thyroid cancer (Compendium of Pharmaceutical and Specialties 35th Edition, 2000). Toxicity caused by gemcitabine hydrochloride includes, for example, hematological toxicity, such as bone marrow suppression; gastrointestinal toxicity, such as nausea, vomiting, and stomatitis; liver toxicity, such as transient increase in serum transaminase; nephrotoxicity, such as proteinuria, hematuria, and hemolysis Uremic syndrome and renal failure; dermatological toxicity, such as rash and alopecia; edema toxicity, such as edema and peripheral edema; and other toxicity, such as fever. This antiproliferative agent has been used to treat pancreatic cancer and non-small cell lung cancer (Compendium of Pharmaceutical and Specialties 35th Edition, 2000).

The present invention includes the prevention or treatment of local cancers or solid tumors. The treatable local cancers or solid tumors include prostate, breast, pancreas, liver, kidney, urogenital system, brain, gastrointestinal system, respiratory system, and head and neck. Localized cancer or solid tumor. The compositions and the like in this article can be used to allow controlled release of purified/modified polytrehalose at a site slightly away from the target tumor, to allow diffusion or even systemic use of an effective concentration of purified/modified polytrehalose Delivery to tumor and/or cancer metastasis to prevent or treat cancer, including cancer metastasis. Some of these cancers are further discussed in the following paragraphs. Prostate cancer

Prostate cancer is a malignant tumor produced in cells that fill the prostate glands. In the United States, it is estimated that 200,000 patients will suffer from prostate cancer this year, and more than 30,000 patients will die from the disease. The proportion of deaths from prostate cancer to new cases is about 15%. Cancer can remain in the prostate, or it can spread to surrounding tissues or distant sites (most commonly lymph nodes and bones). Usually prostate cancer spreads quietly and only produces symptoms when it has progressed beyond the prostate. If prostate cancer has been diagnosed and treated during the early stages, in some studies patients have a 5-year survival rate of 94%.

Prostate cancer is usually described as a disease of men over 50 years of age. In fact, 80% of men with prostate cancer are 60 years old and older. The probability of men diagnosed with prostate cancer during their lifetime is about 1/10, which is about the same as the probability of women suffering from breast cancer. In recent years, the number of reported new cases has increased significantly due to improved tests that can detect the disease early in the disease (usually long before the symptoms appear). The probability of developing prostate cancer in any given year increases with age, but increases significantly after the age of 50.

The current treatment options for prostate cancer depend on the degree of disease progression, the patient's age, and overall health. Elderly patients with only early-stage cancers or affected by additional, more serious diseases can be treated conservatively, however, elderly patients with advanced cancers may experience more aggressive treatment. Prostate cancer has been treated by various methods, including radiation therapy (external beam radiation or brachytherapy), hormonal withdrawal or castration (surgery or chemicals), antiproliferative agents, surgery, and expected treatment (ie, "observation wait"). No treatment guarantees an absolute cure, and some have a large number of side effects.

Early prostate cancer (that is, the tumor is local to the prostate) can be "watched and waited" for treatment. Prostate cancer surgery is recommended for patients whose overall health is otherwise good and whose tumors are limited to prostate glands. A common treatment for localized prostate cancer in men under 70 years of age is radical prostatectomy (ie, surgical removal of the prostate).

Patients whose cancer is local to the prostate area are usually treated with external beam radiation (EBR). Radiation kills cancer cells and shrinks tumors. EBR accounts for less than 20% of local prostate cancer treatment, and about 50% of these patients experience disease recurrence after radiation. Combined with initial prostate cancer detection and increased demand from patients, the use of brachytherapy (ie, localized radiation therapy) has been expected to increase. In 1995, only 2.5% of newly diagnosed patients were treated with brachytherapy. Brachytherapy involves implanting radioactive metal "species" into prostate tumors.

The treatment used for prostate cancer that has spread involves removing testes or hormone therapy. Both are used to inhibit or stop the production of testosterone that has driven cancer growth. About 20% of all prostate cancer patients undergo hormone withdrawal therapy. Hormone therapy includes goserelin acetate (Zoladex®) or leuprolide acetate (Lupron®). Anti-proliferative agents used to treat prostate cancer have included 5-fluorouracil. Breast cancer

In the United States, breast cancer has become the most common cancer among women, with approximately 180,000 new cases diagnosed each year (male breast cancer accounts for approximately 5% of all diagnosed breast cancers). It has become the leading cause of death for women after lung cancer, and it has caused approximately 50,000 deaths each year. American women have 1/8 (or about 13%) chance of developing breast cancer during their lifetime. Over the past decade, the most reported breast cancers are small, primary (independently generated; not caused by cancer metastases) tumors. Approximately 70% to 80% of newly diagnosed patients exhibit early disease (stage 1 or 2), and most of them have not yet involved axillary (under arm) lymph nodes.

Most breast cancers are carcinomas (ie, malignant tumors that grow from epithelial tissue). Less than 1% of breast cancers are sarcomas or tumors caused by connective tissue, bone, muscle or fat. In addition, most breast cancers (approximately 75%) are breast ductal carcinomas, which are produced in tissues alongside breast ducts. A smaller amount of cancer (approximately 7%) is found in the milk leaflets and is called lobular carcinoma. Paget's disease (areola and nipple cancer) and inflammatory carcinomas account for almost all other forms of breast cancer.

Breast cancer treatment is complex and depends on many factors. The two important factors are the type of tumor and the stage of progression. In particular, tumor characteristics help divide individuals into two groups: (1) individuals at low risk of cancer recurrence and (2) individuals at high risk of cancer recurrence. Specific prognostic factors place patients in any of these groups. These factors include the size of the tumor; the presence of the female sex hormones estrogen and progesterone (ER/PR) receptors; the phase of the cell growth cycle (whether the tumor cells are effectively dividing or are in the "S phase"); known as "her-2" -neu protein"; the presence of a protein in the tumor; an indicator of tumor grade, differentiation or change of tumor cells; and tumor ploidy, the number of aggregates of genetic material in tumor cells.

Primary diseases involving no obvious lymph nodes have been treated by mastectomy and radiation therapy. More obvious lymph node involvement can ensure mastectomy and removal of auxiliary lymph nodes. At this stage, the probability of cancer metastasis and local recurrence is already high. The treatment of metastatic disease has been remission, involving radiation therapy and chemotherapy, which is immunosuppression, cytotoxin and leukopenia. Antiproliferative agents including (for example) 5-fluorouracil, cranberry, methotrexate, and paclitaxel have been approved for use against breast cancer. Pancreatic cancer The pancreas is an organ located in the digestive system close to the stomach and small intestine. It has two main functions: production of enzymes and hormones. Pancreatic cancer may appear in the exocrine (ie, enzyme) pancreas (eg, typical pancreatic adenocarcinoma) or may appear in the endocrine (ie, hormone) pancreas.

Exocrine pancreatic cancer is a very serious health problem. In the United States, approximately 28,000 patients are diagnosed with pancreatic cancer, and approximately the same number die from the disease each year. Pancreatic cancer occurs equally in men and women. Due to the difficult diagnosis, the inherent aggressive nature of pancreatic cancer and the scarce available systemic treatment options, after diagnosis, only about 4% of patients diagnosed with pancreatic cancer survive for 5 years. Pancreatic cancer has become the fifth leading cause of cancer death after breast cancer, lung cancer, colon cancer and prostate cancer.

The choice of treatment for pancreatic cancer depends largely on the stage of the tumor. Possible treatments include surgery, antiproliferative agents, radiation, and biological therapy. Surgery has generally been reserved for stage 1 patients whose cancer is considered resectable. Sometimes, a combination of therapy (such as radiation and anti-proliferative agents) given before or after surgery can increase the patient's chance of survival. Antiproliferative agents can be used in clinical trials to treat pancreatic cancer that is considered unresectable (usually stage II or advanced). Antiproliferative agents such as, for example, gemcitabine or 5-fluorouracil have some effects on pancreatic cancer and gemcitabine has been used as a relieving agent. The toxicity caused by these antiproliferative agents is discussed elsewhere in this article. Radiation therapy has some effects on pancreatic cancer when used in combination with chemotherapy. Only radiation therapy can suppress symptoms. This form of treatment has also been used for stage II or advanced pancreatic cancer. Bladder Cancer

In 1998, it is estimated that more than 54,000 new cases of bladder cancer will be diagnosed in the United States and about 15,000 deaths will be attributed to the disease. Bladder cancer has become the fourth most common cancer among American men and the ninth most common cancer among American women. It occurs three times as frequently in men as women. Bladder cancer, mainly a disease of older men, has become a significant cause of disease and death. The risk of bladder cancer increases significantly with age (80% of cases occur in people over the age of 50), and more than one-half of all bladder cancer deaths occur after the age of 70. In white men over 65 years old, the annual disease rate for bladder cancer is already about 2 cases per 1,000 people; this is compared with the ratio of 0.1 cases per 1,000 people under 65 years old. During the life of an individual, the probability of suffering from bladder cancer has been greater than 3%; however, the probability of bladder cancer death has been relatively small (<1%). Bladder cancer rarely occurs in people younger than 40 years old.

Recent studies have shown that certain genes and inherited metabolic capacity can play a role in bladder cancer. Transitional cell carcinoma (TCC) has become the most common form of bladder cancer. TCC usually appears in the form of superficial (surface), papillary (wart-like), exogenous (growing outward) stalks. But in some cases, the TCC may be attached to a broad base or it may present ulcers (within a dent lesion). Papillary TCC usually starts from the proliferating area and then dedifferentiates or loses individual cell characteristics. Only about 10% to 30% of papillary TCCs develop into invasive cancer. In contrast, non-papillary forms of TCC are more likely to become invasive. As mentioned, such TCCs may appear ulcerated or flat. The flat, non-papillary TCC composed of degenerative epithelium has been classified as carcinoma in situ (CIS or TIS). CIS tissue contains larger cells with obvious nucleoli (round bodies in the cell; involved in protein synthesis) and lacking normal polarity.

Treatment of bladder cancer depends on many factors. The most important of these factors is the type of tumor present and its stage. Common treatments include transurethral resection (TUR), electrosurgery, laser surgery, intravesical treatment, antiproliferative agents, surgical therapy, cystectomy, and radiation therapy. Examples of antiproliferative agents used in the treatment of bladder cancer include, for example, 5-fluorouracil, cisplatin, and methotrexate. The toxicity caused by antiproliferative agents, 5-fluorouracil, cisplatin, and methotrexate is discussed elsewhere in this article. Brain cancer

Brain tumors are usually inoperable and more than 80% of patients die within 12 months of diagnosis. In the United States, approximately 18,000 new cases of primary intracranial (brain) cancer are diagnosed each year. This is equivalent to about 2% of all adult cancers. More than 50% of these cancers are highly gliomas ( ie, glioblastoma pleomorphic and degenerative astrocytoma tumors). Patients with these tumors often suffer from severe disabilities, such as motor muscle dysfunction, epilepsy, and visual abnormalities.

Tumors that start in brain tissue are called primary brain tumors. Primary brain tumors are classified by the type of tissue in which they started. The most common brain tumor is glioma, which starts in glial (supportive) tissue. Others include astrocytoma, brainstem glioma, ependymoma, and oligodendroglioma.

Surgical removal of brain tumors has been recommended for most types and most locations and should be done as far as possible within the limits of retaining nerve function. The exception to this rule is deep tumors, such as pontine gliomas, which are diagnosed on clinical signs and treated for approximately 50% of the time without initial surgery. However, in many cases, a biopsy diagnosis is performed. Stereotactic biopsy can be used for lesions that are difficult to reach and excise. Rarely curable or unresectable patients with brain tumors should be considered clinical trials for the evaluation of radiation sensitizers, high temperature or interstitial brachytherapy combined with external beam radiation therapy to improve local control of tumors Or candidates for research in evaluating new drugs and biological response modifiers.

Radiation therapy has a major role in the treatment of most tumor types and can increase the cure rate or prolong disease-free survival. Radiation therapy can also be used to treat relapses in patients who were initially treated only by surgery. Chemotherapy can be used before, during, or after surgery and radiation therapy. Chemotherapy is also used to treat recurrent tumors. Antiproliferative agents used to treat brain cancer include cisplatin. Examples of toxicity associated with this anti-proliferative agent are discussed elsewhere in this article. Restenosis

Restenosis is a form of chronic vascular injury that causes thickening of blood vessel walls and loss of blood flow supplied by blood vessels to tissues. This inflammatory disease can arise in response to a blood vessel rebuilding procedure that includes any operation to reduce blood vessel blockage. Therefore, restenosis has become the main limiting factor limiting the effectiveness of these procedures.

The present discussion includes, for example, the prevention or treatment of restenosis by administering a therapeutically effective amount of a combination of an oligonucleotide therapeutic agent and an anti-inflammatory agent to blood vessels. Suitable compositions include a polymeric carrier that can be surgically implanted at a restenosis or potential restenosis site or can be injected via a catheter in the form of a polymerizable paste or gel. Suitable compositions can include the purified/modified polytrehalose discussed herein. arthritis

Rheumatoid arthritis (RA) is a debilitating chronic inflammatory disease characterized by joint tissue pain, swelling, synovial cell proliferation (pannus formation) and damage. In the advanced stages, the disease usually damages critical organs and can be fatal. The disease involves multiple members of the immune system (macrophages/monocytes, neutrophils, B cells, and T cells), complex interleukin interactions, and synovial cell dysfunction and proliferation. It has been suggested to use disease-modifying anti-rheumatic drugs (DMARD), such as methotrexate, for early invasive treatment, which is discussed elsewhere in this article.

Lens-induced arthritis is characterized by the activation of lens-induced macrophages and neutrophils in the joints followed by severe pain lasting several days. Disease progression makes the interval between attacks shorter and the patient's morbidity increases. The disease has been largely symptomatically treated with non-steroidal anti-inflammatory drugs (NSAIDs), such as diclofenac sodium (Voltaren®). This anti-inflammatory agent is toxic and includes central nervous system toxicity such as dizziness and headache; dermatological toxicity such as rash and pruritus; gastrointestinal toxicity such as exacerbated ulcerative colitis and Crohn's disease; Urogenital toxicity, such as acute renal failure and renal papillary necrosis; blood toxicity, such as granulocyte deficiency, leukopenia, and thrombocytopenia; liver toxicity, such as elevated liver transaminase and hepatitis; and other toxicity, such as asthma and Systemic allergic reactions.

The discussion of the present invention includes the prevention or treatment of rheumatoid arthritis, for example, by administering to the patient a therapeutically effective amount of an oligonucleotide therapeutic agent and optionally an anti-inflammatory agent. Suitable compositions include polymeric carriers, which can be in the form of anti-inflammatory agents and controlled release carriers of microparticles in the form of controlled release carriers of oligonucleotide therapeutic agents (which in turn have been incorporated into polymeric carriers Middle) Injection into the joint. Suitable compositions can include the purified/modified polytrehalose discussed herein. Such polymerizable carriers can take the form of polymerizable microspheres, pastes, or gels. Inflammatory pathology

The composition and the like herein may optionally include, for example, administering a composition containing an oligonucleotide therapeutic agent and an anti-inflammatory agent to a patient to suppress or treat an inflammatory condition involving neutrophils. Examples of such conditions include crystal-induced arthritis; osteoarthritis; non-rheumatic inflammatory arthritis; mixed connective tissue disease; Sjogren's syndrome; ankylosing spondylitis; Behcet Syndrome (Behget's syndrome); Sarcoma; Psoriasis; Eczema; Inflammatory bowel disease; Chronic inflammatory lung disease; Neurological disorders and multiple sclerosis. Some of these diseases are discussed further in the following paragraphs. Chronic inflammatory skin diseases ( including psoriasis and eczema )

Psoriasis is a common chronic inflammatory skin disease characterized by itching, burning, stinging, protruding, thickening, and scaly lesions that are prone to bleeding. When these diseases have cell proliferation and angiogenesis components in the late stages of the disease, patients usually have accompanying arthritis symptoms. Symptoms can be treated with steroidal anti-inflammatory agents such as prednisone or anti-proliferative agents such as methotrexate, which are discussed elsewhere in this article. The compositions herein can also be used to inhibit or otherwise treat and/or prevent chronic inflammatory skin diseases, such as psoriasis and/or eczema.

The following provides some additional representative examples of inflammatory diseases that can be treated by the compositions discussed herein, including, for example, arterial embolization of arteriovenous malformations (vascular malformations); excessive menstruation; acute bleeding; central nervous system disorders and splenic function Hyperactivity; inflammatory skin diseases, such as psoriasis; eczema diseases (atopic dermatitis, contact dermatitis, eczema); immunobullous disease; and inflammatory arthritis including various pathologies, These conditions include rheumatoid arthritis, mixed connective tissue disease, Sjogren's syndrome, ankylosing spondylitis, Behcet's syndrome, sarcomatosis, crystal-induced arthritis and osteoarthritis (all of the above features Inflamed and painful joints are important symptoms). Ischemia

Ischemia or ischemia involves the limitation of blood supply sources, which may include insufficient supply of oxygen, glucose and other components required for proper tissue function, resulting in tissue damage and/or dysfunction. Ischemia can cause serious problems. For example, tissue can become hypoxic, necrotic, and can form clumps. Various attempts have been made to prevent and/or treat ischemia. For example, restore blood flow or reperfusion. However, restoring blood involves the reintroduction of oxygen, which can cause additional damage due to the production of free radicals, which in turn can cause reperfusion injury. Reperfusion injury can cause serious problems. The compositions herein can be used to inhibit or otherwise treat and/or prevent ischemia and/or reperfusion injury. Endotoxemia

Endotoxemia is the presence of endotoxin in the blood. Endotoxemia can cause serious problems. For example, endotoxemia can cause septic shock. The compositions herein can be used to inhibit or otherwise treat and/or prevent endotoxemia. Keloid scarring

The nature of keloids causes wounds to heal with raised scars. Protruding scars of keloid traits involve abnormal fibrous scarring. The nature of keloids causes serious problems such as pain and appearance damage. The compositions herein can be used to inhibit or otherwise treat and/or prevent keloid traits and the resulting raised scars.

Keloids (keloid scars) are scar types that grow and expand beyond normal skin. Keloids involve abnormal collagen growth, including abnormal growth of type I and type III collagen. Keloids cause serious problems such as pain, itching, and if infected, they can ulcerate. Attempts have been made to treat or prevent keloids, including the use of surgery, dressings, steroid injections, and laser therapy. The compositions herein can be used to inhibit or otherwise treat and/or prevent keloids. dermatitis

Dermatitis includes inflammation of the skin, including atopic dermatitis and contact dermatitis. For example, contact dermatitis involves local rashes and/or irritation of the skin after the skin comes into contact with foreign substances. For example, atopic dermatitis is a long-term recurrent, itchy skin disease. Atopic dermatitis is sometimes referred to as prurigo Besnier, neurodermatitis, endogenous eczema, flexed eczema, infant eczema, eczema in children, and itching (prurigo diathsique) . Eczema is a disease in the form of dermatitis. Other types of dermatitis include spongiotic dermatitis, seborrheic dermatitis (dandruff), dyshidrotic dermatitis (sweat herpes), rubella, vesicular dermatitis (bulbar dermatitis) and epidemics Rubella. Dermatitis can cause serious problems. For example, dry skin, skin rashes, skin edema, skin redness, itching, skin scabs, cracking, blistering, exudation, and bleeding. Attempts have been made to treat or prevent dermatitis, including the use of corticosteroids and coal tar. The composition herein can be used to inhibit or otherwise treat and/or prevent dermatitis, including atopic dermatitis, eczema, contact dermatitis, cavernous dermatitis, seborrheic dermatitis, sweating dermatitis, rubella, Vesicular dermatitis and epidemic rubella. Rosacea

Rosacea is a chronic disease or condition typically characterized by facial erythema. Rosacea can cause serious problems. For example, rosacea can cause additional symptoms, including capillary dilatation, papules, pustules, pain sensation, and in advanced cases, rhinophyma (red lobulated nose) may occur. Subtypes of rosacea include lupus erythematosus, pyogenic papular rosacea, lumpy rosacea and ocular rosacea. Attempts have been made to treat or prevent rosacea, including the use of non-steroidal anti-inflammatory drugs and antibiotics. The compositions herein can be used to inhibit or otherwise treat and/or prevent rosacea, including its lupus erythematosus, pustular papule-like, rosacea, and ocular subtypes. Medical devices, medical materials, combinations and pharmaceutical products

The discussion herein also provides medical devices, combinations, and medical products, which comprise the composition as discussed herein in a medical device, combination product, or pharmaceutically acceptable container. The product may also include a notice connected to the container, typically in the form prescribed by the regulatory agency that manages the manufacture, use, or sale of medical devices, combinations, and pharmaceutical or biological agents, so that the notice reflects the composition’s approval by the agency , Such as purified/modified polytrehalose has been approved as, for example, for human or veterinary medical administration to treat proliferative or inflammatory diseases (such as (for example) inflammatory arthritis, restenosis, surgical adhesions, psoriasis and peritonitis) Notice of anti-proliferative agent or anti-inflammatory agent. Instructions for using the purified/modified polytrehalose herein can also be included. Such instructions may include information about the patient's administration and mode of administration.

The present application further relates to various elements involved in making the purified/modified polytrehalose, systems, etc. discussed herein, including methods of making the composition itself and methods of use thereof, including (for example) treating Symptoms, diseases, etc.

This application further includes medical devices, medical materials, medical combination products, and pharmaceutical products for the treatment of fibrous adhesions, arthritis, psoriasis, or other diseases as needed, which includes the purified/modified polytrehalose and Polytrehalose composition. These materials can be used to treat fibrous adhesions, such as surgical adhesions, arthritis, psoriasis, or other diseases as needed. Also provided are methods of manufacturing and using such pharmaceutical products that are capable of reducing symptoms associated with at least one of fibrous adhesions, arthritis, and psoriasis in patients (including human patients), such methods include pharmacologically effective The amount of polytrehalose (such as fucoidan as discussed herein) is combined with a pharmaceutically acceptable excipient or buffer.

The following examples provide illustrative discussion of certain embodiments herein, but the scope of the invention and patent applications are not limited thereto. Example 1 : Chemical structural modification

The exudate extract was obtained from Laminaria Hyperborea . The exudate extract was filtered through tangential flow filtration (TFF) through a 100 kDa filter to remove small molecules. Samples of the resulting retentate were lyophilized to obtain sample A otherwise unmodified. The resulting retentate reached 0.25 M NaOH by adding 10 M NaOH solution and standing at room temperature for 16 hours. The resulting sample was then centrifugally filtered through a 50 kDa filter and the resulting retentate was collected and lyophilized to obtain alkali-treated sample B. Both unmodified sample A and base-treated sample B were analyzed by proton nuclear magnetic resonance spectroscopy ( ¹ H-NMR) and the resulting ¹ H-NMR spectrum is shown in FIG. 2A .

Figure 2A shows the chemical structural modification of polytrehalose: the wider peak with a chemical shift of about 2.0 ppm present in unmodified sample A does not exist in sample B treated with alkali.

The unmodified sample A and the alkali-treated/modified sample B were further analyzed by 2D ¹ H- ¹³ C heteronuclear multiple quantum coherence (HMQC). The HMQC spectrum shown in FIG. 2B was obtained on a 600 MHz spectrometer equipped with a 5-mm cold probe with solvent signal suppression at 70°C. A large number of scans of the HMQC spectrum are obtained each time in 8 increments of 256-512 scans in the carbon size range of 10-30 ppm; such scans are combined to produce the spectrum in Figure 2B .

The HMQC spectrum of unmodified sample A has a cross peak corresponding to O-acetyl, which is indicated by the circled signal in FIG. 2B . This cross peak does not exist in the spectrum of sample B after alkali treatment. This demonstrates the removal of acetyl groups from polytrehalose, and therefore the chemical structural modification of polytrehalose in alkali-treated sample B by NaOH treatment. Example 2 : Physically induced flocculation

The brown powder raw material fucoidan was dissolved in distilled water at about 10% w/v to obtain a starting solution. Sodium chloride was added to the starting solution to produce a mixture with a final sodium chloride concentration of about 0.1 M. Heat the mixture to near boiling for 10-15 minutes. Treatment of the mixture at this temperature induces flocculation of suspended impurities and particulate non-fucoidan materials. The mixture was centrifuged at 2300 gravity for 40 minutes to separate the fucoidan-containing solution from the flocculated non-fucoidan component. The solution containing fucoidan was visually inspected and a visible reduction in particulate matter and color was observed. The lyophilized portion of the solution containing fucoidan contains an off-white powder with a significantly lighter color than the raw material fucoidan used. The UV/Vis spectrum of the raw fucoidan at 10 mg/mL in water (UV/Vis spectrum) and the purified/modified polytrehalose at 10 mg/mL in water can be obtained, for example : Determine the total absorbance in the visible region of the spectrum, which is between about 400nm and about 700nm; and observe that the total absorbance in the purified/modified polytrehalose is reduced by at least 5%, 10% relative to the raw fucoidan Or 20% to quantify and compare color loss. Example 3 : Solid phase extraction

Fucoidan, a raw material of brown powder, was added to a mixture of 0.5 M NaOH in 70% v/v ethanol/water at 40 degrees Celsius. The resulting reaction mixture was stirred and kept at 40 degrees Celsius for 2 hours. The reaction mixture was then centrifuged to separate the solid purified/modified fucoidan from the 70% v/v ethanol/water supernatant containing 0.5 M NaOH containing extracted impurities.

At the time of observation, for the human eye, it was found that the solid purified/modified fucoidan contained a significantly lighter color than the raw material fucoidan. This loss of color indicates the removal of impurities (such as brown algae polyphenols), since polytrehalose does not contain chromophores and therefore will be colorless when completely pure. The UV/Vis spectrum of the raw fucoidan at 10 mg/mL in water (UV/Vis spectrum) and the purified/modified polytrehalose at 10 mg/mL in water can be obtained, for example : Determine the total absorbance in the visible region of the spectrum, which is between about 400nm and about 700nm; and observe that the total absorbance in the purified/modified polytrehalose is reduced by at least 5%, 10% relative to the raw fucoidan Or 20% to quantify and compare color loss. Example 4 : Chemically induced precipitation

The raw material fucoidan composition was dissolved in distilled water at 15% w/v to form a starting solution. It was found through observation that the starting solution contained suspended particles. Calcium chloride was added to the starting solution to a level of 0.5 M to produce a reaction mixture. To simulate natural fucoidan with known impurities, sodium alginate was added at a concentration of 5% w/w alginate/fucoidan and starch was added at a concentration of 5% w/w starch/fucoidan. In this case, starch is used as a simulant for laminose. 10 M NaOH was added dropwise to the reaction mixture to bring the pH between 7 and 8. This is done to avoid degradation of fucoidan in the reaction mixture. The minimum amount of 10 M NaOH was also added to the reaction mixture to avoid acidification of the reaction mixture due to the subsequent addition of phosphoric acid. The reaction mixture was introduced into 0.5 M phosphate via addition of phosphoric acid. This causes the flocculation of suspended particles and sediment impurities through the action of calcium phosphate, which is formed by the reaction of calcium chloride and phosphoric acid. The reaction mixture was allowed to stand at room temperature for 10 minutes to allow flocculation to continue. The reaction mixture was centrifuged at 17568 gravity for 17 minutes to separate the supernatant solution containing the desired purified fucoidan from the flocculated impurities. The supernatant solution was visually inspected to qualitatively evaluate color and particle removal. An aliquot supernatant solution is also analyzed by UV/Vis absorption in the 300-800 nm region to evaluate the non-fucoidan component that scatters light and/or absorbs light in the UV/Vis spectral region Removal. An aliquot of the supernatant solution was also lyophilized to obtain polytrehalose content. An aliquot of the supernatant solution was also hydrolyzed in 3M HCl at 90 degrees Celsius and analyzed by High Performance Anion Exchange-Pulsed Amperometry Detection (HPAE-PAD) to detect the total Carbohydrates and assess the removal of laminose and alginate. The impurities were quantified relative to the monomeric glucose standard to assess the removal of cumulose and monomeric mannonic acid and monomeric guluronic acid, and then to the removal of alginate.

*：經由代表性起始褐藻糖膠測定之值表 1 ：起始褐藻糖膠及經處理溶液之分析結果實施例 5 ：化學誘發之沈澱 The analysis results of the starting fucoidan and the resulting purified/modified polytrehalose are presented in Table 1 below.

*: Values measured by representative starting fucoidan Table 1 : Analysis results of starting fucoidan and treated solution Example 5 : Chemically induced precipitation

The raw material fucoidan composition was dissolved in distilled water at 15% w/v to form a starting solution. It was found through observation that the starting solution contained suspended particles. To simulate natural fucoidan with known impurities, sodium alginate was added at a concentration of 5% w/w alginate/fucoidan and starch was added at a concentration of 5% w/w starch/fucoidan. In this case, starch is used as a simulant for laminose. 10 M NaOH was added dropwise to the starting solution to bring the pH between 7 and 8. This operation is performed to avoid the degradation of fucoidan in the solution when the subsequent addition of aluminum sulfate causes the starting solution to be acidic. The starting solution was introduced into 0.1 M aluminum sulfate to produce a reaction mixture. This starts the precipitation of impurities and the flocculation of impurities and suspended particles by the aluminum hydroxide formed at the same time. The reaction mixture was allowed to stand at room temperature for 10 minutes to allow flocculation to continue. The reaction mixture was centrifuged at 17568 gravity for 17 minutes to separate the supernatant solution containing the desired purified fucoidan from the flocculated impurities. The supernatant solution was visually inspected to qualitatively evaluate color and particle removal. An aliquot supernatant solution is also analyzed by UV/Vis absorption in the 300-800 nm region to evaluate the non-fucoidan component that scatters light and/or absorbs light in the UV/Vis spectral region Removal. An aliquot of the supernatant solution was also lyophilized to obtain polytrehalose content. An aliquot of the supernatant solution was also hydrolyzed in 3M HCl at 90 degrees Celsius and analyzed by high-efficiency anion exchange-pulsed amperometric detection analysis to detect total carbohydrates and evaluate the removal of laminose and alginate. The impurities are quantified relative to the monomeric glucose standard to assess the removal of laminose and monomeric mannonic acid and monomeric gulonic acid, and in turn to evaluate the removal of alginate.

*：經由代表性起始褐藻糖膠測定之值表 2 ：起始褐藻糖膠及經處理溶液之分析結果實施例 6 ：化學誘發之沈澱 The analysis results of the starting fucoidan and the resulting purified/modified polytrehalose are presented in Table 2 below.

*: Values measured through representative starting fucoidan Table 2 : Analysis results of starting fucoidan and treated solution Example 6 : Chemically induced precipitation

The starting fucoidan composition was dissolved in distilled water at 15% w/v to form a starting solution. It was found through observation that the starting solution contained suspended particles. To simulate natural fucoidan with known impurities, sodium alginate was added at a concentration of 5% w/w alginate/fucoidan and starch was added at a concentration of 5% w/w starch/fucoidan. In this case, starch is used as a simulant for laminose. Calcium chloride was added to the starting solution to a level of 0.5 M to produce a reaction mixture. This triggers the precipitation of alginate. 10 M NaOH was added dropwise to the reaction mixture to bring the pH between 7 and 8. This is done to avoid degradation of fucoidan in the reaction mixture. The reaction mixture was introduced into 0.5 M aluminum sulfate. This calcium sulfate is formed by the action of calcium sulfate and aluminum hydroxide to cause the flocculation of suspended particles, calcium alginate precipitation and other impurities. The calcium sulfate is formed by the reaction of calcium chloride and aluminum sulfate. The aluminum hydroxide is It is formed from a reaction mixture containing aluminum sulfate. The reaction mixture was allowed to stand at room temperature for 10 minutes to allow flocculation to continue. The reaction mixture was centrifuged at 17568 g for 17 minutes to separate the supernatant solution containing the desired purified fucoidan from the flocculated impurities. The supernatant solution was visually inspected to qualitatively evaluate color and particle removal. An aliquot supernatant solution is also analyzed by UV/Vis absorption in the 300-800 nm region to evaluate the non-fucoidan component that scatters light and/or absorbs light in the UV/Vis spectral region Removal. An aliquot of the supernatant solution was also lyophilized to obtain polytrehalose content. An aliquot of the supernatant solution was also hydrolyzed in 3M HCl at 90 degrees Celsius and analyzed by high-efficiency anion exchange-pulsed amperometric detection analysis to detect total carbohydrates and evaluate the removal of laminose and alginate. The impurities are quantified relative to the monomeric glucose standard to assess the removal of laminose and monomeric mannonic acid and monomeric gulonic acid, and in turn to evaluate the removal of alginate.

*：經由代表性起始褐藻糖膠測定之值表 3 ：起始褐藻糖膠及經處理溶液之分析結果實施例 7 ：液 - 液萃取 The analysis results of the starting fucoidan and the resulting purified/modified polytrehalose are presented in Table 3 below.

*: Values measured through representative starting fucoidan Table 3 : Analysis results of starting fucoidan and treated solution Example 7 : Liquid - liquid extraction

The starting polytrehalose composition containing impurities was dissolved in distilled water at 10 mg/mL to produce an aqueous starting solution. 20% v/v heptane was added to the aqueous starting solution containing the starting fucoidan composition and the organic aqueous mixture was then mixed under higher shear stress for 30 minutes. The mixing was terminated and the organic aqueous mixture was placed in a separatory funnel to separate the organic phase from the aqueous phase. The denser aqueous phase containing the desired polytrehalose component is settled to the bottom of the separatory funnel, while the less dense organic phase containing impurities is present at the upper end of the separatory funnel. The organic aqueous mixture was allowed to stand in a separatory funnel for 10 minutes. The aqueous phase is then decanted and the desired purified/modified polytrehalose in solution is collected. Compared to the starting polytrehalose composition, it can be found that the purified/modified polytrehalose in solution contains less lipid, fatty acid, brown algae between about 30%, 50%, 70% to about 100% Polyphenols, proteins, fucoxanthin and/or chlorophyll. Example 8 : Liquid - liquid extraction

The starting polytrehalose composition containing impurities was dissolved in distilled water at 10 mg/mL to produce an aqueous starting solution. 20% v/v 1-butanol was added to the aqueous starting solution containing the starting fucoidan composition and then the organic aqueous mixture was mixed under higher shear stress for 30 minutes. The mixing was terminated and the organic aqueous mixture was placed in a separatory funnel to separate the organic phase from the aqueous phase. The denser aqueous phase containing the desired polytrehalose component is settled to the bottom of the separatory funnel, while the less dense organic phase containing impurities is present at the upper end of the separatory funnel. The organic aqueous mixture was allowed to stand in a separatory funnel for 10 minutes. The aqueous phase is then decanted and the desired purified/modified polytrehalose in solution is collected. Compared to the starting polytrehalose composition, it can be found that the purified/modified polytrehalose in solution contains less lipid, fatty acid, brown algae between about 30%, 50%, 70% to about 100% Polyphenols, proteins, fucoxanthin and/or chlorophyll. Example 9 : Liquid - liquid extraction

The starting polytrehalose composition containing impurities was dissolved in distilled water at 10 mg/mL to produce an aqueous starting solution. 20% v/v ethyl acetate was added to the aqueous starting solution containing the starting fucoidan composition and then the organic aqueous mixture was mixed under higher shear stress for 30 minutes. The mixing was terminated and the organic aqueous mixture was placed in a separatory funnel to separate the organic phase from the aqueous phase. The denser aqueous phase containing the desired polytrehalose component is settled to the bottom of the separatory funnel, while the less dense organic phase containing impurities is present at the upper end of the separatory funnel. The organic aqueous mixture was allowed to stand in a separatory funnel for 10 minutes. The aqueous phase is then decanted and the desired purified/modified polytrehalose in solution is collected. Compared to the starting polytrehalose composition, it can be found that the purified/modified polytrehalose in solution contains less lipid, fatty acid, brown algae between about 30%, 50%, 70% to about 100% Polyphenols, proteins, fucoxanthin and/or chlorophyll. Example 10 : Diafiltration

表 4 ：起始褐藻糖膠及經處理溶液之分析結果實施例 11 ： 超臨界流體萃取 Provide a starting solution or starting fucoidan composition containing about 8% w/v. The starting solution was filtered through a 0.22 micron filter. Aliquot of the sample was filtered through cation content of the starting solution based analysis by inductively coupled plasma mass spectrometry (ICP-MS) was measured and was found to contain greater than 0.01% w / w aluminum / polyethylene trehalose, higher than 10 ^{- 5} % w/w arsenic/polytrehalose and higher than 0.01% w/w calcium/polytrehalose are all undesirable levels for each corresponding cation. The starting solutions of 4 diafiltration volumes were diafiltered by 0.1 M EDTA, 0.01 M NaOH solution. The resulting retained fucoidan solution was then diafiltered by about 2.5 diafiltration volumes of 5 mM Na2SO3, 5 mM NaCl solution. The cation content of the second retained fucoidan solution was analyzed by ICP-MS. The starting fucoidan composition and the results of the purified/modified polytrehalose obtained are shown in Table 4 below.

Table 4 : Analysis results of starting fucoidan and treated solution Example 11 : Supercritical fluid extraction

Provide about 100 g of solid starting fucoidan composition. Place the solid in the supercritical extractor. The extractor was pressurized to 5800 psi and heated to 50 degrees Celsius, and then purged with supercritical carbon dioxide at 100 mL/min for 3 hours. The supercritical carbon dioxide is removed from the extractor and the solid modified/purified fucoidan is collected and analyzed for impurities. Compared to the starting fucoidan composition, it can be found that the collected solid modified/purified fucoidan contains between about 30% and about 100% less lipid, fatty acids, fucoidan, laminose, seaweed Acid salts, proteins, Mena reaction products, fucoxanthin, chlorophyll, free ions, bacteria and/or DNA. Example 12 : Chemically induced precipitation, lysis and flocculation

It was found that the starting fucoidan composition containing about 0.70% w/w total nitrogen was dissolved in distilled water at 15% w/v to form a starting solution. The presence of total nitrogen indicates the presence of undesirable impurities, such as cellular components, DNA, proteins, and bacteria. The reduction in total nitrogen indicates that these nitrogen-containing impurities have been removed from the starting fucoidan composition or from the fucoidan polymer, as the case may be, because the nitrogen-containing impurities can be chemically or ionicly combined For such fucoidan molecules.

It was found through observation that the starting solution contained suspended particles. Calcium chloride was added to the starting solution to a level of 0.5 M to produce a reaction mixture. This operation caused the precipitation of the solid part suspected of containing impurities. About 15 mL of 10 M NaOH was added dropwise to the reaction mixture to bring the pH between 7 and 8. This is done to avoid degradation of fucoidan in the reaction mixture. The reaction mixture was introduced into 0.5 M phosphate via addition of phosphoric acid. This causes the flocculation of suspended particles and sediment impurities through the action of calcium phosphate, which is formed by the reaction of calcium chloride and phosphoric acid. The reaction mixture was centrifuged at 33,746 gravity for 5 minutes to separate the first purified/modified fucoidan-containing supernatant solution from the flocculated impurities. The first purified/modified fucoidan was found to contain about 0.10% w/w total nitrogen. A portion of the supernatant solution containing the first purified/modified fucoidan was further purified by diafiltration through a 100 kDa MWCO centrifugal filter against 6 diafiltration volumes of 5 mM NaCl. The resulting first retained purified/modified fucoidan was found to contain about 0.08% w/w total nitrogen.

The second part of the first purified/modified fucoidan was further processed by adding 1 M sodium dodecyl sulfate solution as a cell disrupting agent to a concentration of 0.010 M. Add 10 M NaOH solution to a concentration of 0.26 M to make the mixture alkaline. The resulting reaction mixture was stirred at room temperature for about 30 minutes to obtain a cloudy light brown mixture.

After about 30 minutes, 45% w/v KOH solution was added to reach a concentration of about 0.04 M. The addition of potassium causes the precipitation of SDS and undesired impurities together with SDS. Add 48% w/v aluminum sulfate solution to a concentration of about 0.06 M. The formation of aluminum hydroxide flocculates undesired impurities in the reaction mixture. The sodium sulfite solid is added and dissolved to a concentration of 0.02 M to stop the potential oxidant in the reaction mixture.

The resulting reaction mixture was stored in the refrigerator for approximately 16 hours, followed by centrifugation at 33,746 gravity for 5 minutes to separate the second purified/modified fucoidan-containing supernatant solution from the flocculated impurities. The second purified/modified fucoidan was found to contain about 0.06% w/w total nitrogen. A portion of the second purified/modified fucoidan was further processed by diafiltration through a 100 kDa MWCO centrifugal filter against 6 diafiltration volumes of 5 mM NaCl to obtain a second retained purified/modified fucoidan glue. The resulting second retained purified/modified fucoidan was found to contain about 0.03% w/w total nitrogen. Example 13 : Preparation of five purified / modified polytrehalose

The methods discussed herein can be used, combined, modified, and arranged in any way to obtain purified/modified polytrehalose. Use the combination of chemically induced precipitation and diafiltration discussed in Examples 4, 5, 6, and 10 to prepare five portions of purified/modified polytrehalose to evaluate the use of purified/modified polytrehalose in medical and surgical applications In the effect. These 5 parts of polytrehalose are referred to herein as polytrehalose 1 to polytrehalose 5. The methods discussed in Example 4 and Example 10 were used to produce polytrehalose 1 and polytrehalose 2 on a scale of about 2 kg. The methods discussed in Example 4 and Example 10 were used to produce polytrehalose 3 and polytrehalose 5 on a scale of about 30 g. The method discussed in Example 5 and Example 10 was used to produce polytrehalose 4 on a scale of about 1 kg. The polytrehalose 1 to 5 were converted to solid purified/modified polytrehalose by diafiltration against a low conductivity salt solution, followed by lyophilization to obtain a white solid. Two additional poly-trehaloses are extracted from brown seaweed, referred to herein as poly-trehalose 6 and poly-trehalose 7. With FMC BioPolymer®, polytrehalose 6 was provided as a solid composition. None of the methods described above are used to produce polytrehalose 6. Polytrehalose 7 was extracted from brown seaweed by near boiling HCl. By using ethanol as a precipitating agent to selectively precipitate to remove some impurities. After selective precipitation, polytrehalose was collected as a solid composition by further precipitating polytrehalose with ethanol, centrifugation and lyophilization. Polytrehalose 7 was further processed by the method discussed in Example 3, then dissolved in water and diafiltered against deionized water, and then lyophilized to obtain polytrehalose 7 as a solid composition. As discussed in Example 14 and Example 15 below, the fucose, galactose, sulfate and total relative ion content of polytrehalose 1 to polytrehalose 7 were determined. These polytrehaloses 1 to 7 are discussed further below, for example in Example 16 and Table 6. Example 14 : Measuring the corrected fucose content and corrected galactose content of polytrehalose 1 to polytrehalose 7

The solid polytrehalose composition was dissolved in 72% w/w sulfuric acid at 40 mg/mL and incubated at 45° C. for 30 minutes in a water bath. The acidic hydrolysate was then diluted to 4% w/w sulfuric acid in a high-pressure test tube and incubated at 120°C for 60 minutes. The obtained second acidic hydrolysate was diluted to 1/333 concentration with distilled water and flowed on a high-efficiency anion exchange column chromatography setup (HPAE-PAD) with a pulsed amperometric detector. Analyte separation was achieved by using a gradient-free pump to flow 10 mM NaOH eluent at 1.0 mL/min.

The uncorrected fucose content of polytrehalose was determined by interpolation of the standard curve on fucose. The uncorrected galactose content of polytrehalose was determined by standard addition methods. The corrected fucose content was determined by considering the addition of one water molecule after hydrolyzing glycosidic bonds and considering adding two hydroxyl groups after each fucose hydrolyzed two sulfate bonds. Determine the corrected galactose content by considering adding a water molecule after hydrolyzing the glycosidic bond.

The results of this analysis are shown in Table 5 below. Example 15 : Measuring the total sulfate content, total relative ion content and total water content of polytrehalose 1 to polytrehalose 7

The solid polytrehalose composition was dissolved in deionized water, hydrolyzed under acidic conditions and analyzed by ICP-MS for% w/w total sulfur and relative ion content. The sulfur content is converted to the sulfate content by multiplying the sulfur content by the molar ratio of sulfate to sulfur to obtain the% w/w sulfate content of the purified/modified polytrehalose. The relative ions observed in the purified/modified polytrehalose discussed herein include potassium and sodium relative ions. The results of this analysis are shown in Table 5 below.

表 5 ：聚海藻糖之呈% w/w形式之組分Table 5 below also shows the% w/w results for total corrected fucose, corrected galactose, and sulfate. The results of determining the total fucose, galactose, and sulfate by adding the corrected fucose, corrected galactose, and sulfate values are shown in Equation 1 below including the aspects discussed above For more detailed and complete calculations. The total relative ion content is determined by adding the total sodium and total potassium content together. Equation 1 :

Table 5 : Components of polytrehalose in the form of% w/w

Table 5 demonstrates that less than about 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% w/w impurities can be prepared using the methods discussed herein Purified/modified polytrehalose.

Table 5 further demonstrates that purified/modified polytrehalose has been produced with total galactose, fucose, and sulfate content between about 77% w/w and about 87% w/w.

Table 5 further demonstrates purified/modified polytrehalose with a total relative ion content between about 9% w/w and about 14% w/w of polytrehalose.

The total water content of poly-trehalose 1, poly-trehalose 3, poly-trehalose 4, and poly-trehalose 5 was determined by loss-of-drying (LOD) at 104°C. The total water content was determined to be 3.8%, 2.4%, 3.2% and 4.7% w/w of the corresponding purified/modified polytrehalose. Example 16 : Measuring the molecular weight distribution of polytrehalose 3 and polytrehalose 4

Gel permeation chromatography (GPC) is used to evaluate the molecular weight distribution obtained for purified/modified polytrehalose: polytrehalose 3 and polytrehalose 4. There are a large number of different parameters, columns, and standards that can be used in gel permeation chromatography, making various instrument settings available for molecular weight analysis. For the molecular weight determination in this article, the following parameters were used for GPC: The mobile phase was 0.1 M sodium nitrate flowing at 0.6 mL/min. The column chamber and detector are at 30°C. Waters 2414 refractive index detector is used for detection.

Suitable GPC columns include GPC columns compatible with aqueous solvents, such as columns packed with at least one of the following: sulfonated styrene-divinylbenzene, NH functionalized acrylate copolymer network, modified Silicon dioxide and gel based on hydroxylated polymethacrylate. For the analysis in this article, three columns are used in series, which contains a 40 mm long protective column with an inner diameter (ID) of 6 mm, filled with a 6 pm particle size based hydroxylated polymethacrylate gel; It is the first 300 mm analytical GPC column with a 7.8 mm ID, packed with a 12 pm particle size hydroxylated polymethacrylate based gel, which has a discharge limit of about 7,000 kDa and between about 50 kDa and about 5,000 kDa Effective molecular weight range between; followed by a second 300 mm analytical GPC column with a 7.8 mm ID, filled with a 10 pm particle size based hydroxylated polymethacrylate gel, which has a discharge limit of about 7,000 kDa and An effective molecular weight range between about 1 kDa and about 6,000 kDa. The total effective molecular weight range of the column setup is between about 1 kDa and about 6,000 kDa. An example of this string arrangement can be an Ultrahydrogel® protection-Ultrahydrogel® 2000-Ultrahydrogel® linear string connected in series.

Quantify sample flow relative to a standard curve containing traceable standards from American Polymer Standards: DXT3755K (peak molecular weight = 2164 kDa), DXT820K (peak molecular weight = 745 kDa), DXT760K (peak molecular weight = 621 kDa), DXT670K ( Peak molecular weight = 401 kDa), DXT530K (peak molecular weight = 490 kDa), DXT500K (peak molecular weight = 390 kDa), DXT270K (peak molecular weight = 196 kDa), DXT225K (peak molecular weight = 213 kDa), DXT150K (peak molecular weight = 124 kDa) ), DXT55K (peak molecular weight = 50 kDa), DXT50K (peak molecular weight = 44 kDa) and DXT5K (peak molecular weight = 4 kDa), the peak molecular weight of these standards is between about 4 kDa and about 2,200 kDa. The standard curve used may include, for example, at least one of Dextran 3755 kDa, Dextran 50 kDa and Dexran 55 kDa and between 3 and 6 additional traceable standards discussed herein, the calibration point is the peak value of the calibrator used Molecular weight. An example calibration curve may consist of the following: DXT3755K, DXT 820K, DXT530K, DXT500K, DXT225K and DXT55K. The column used herein has a total effective molecular weight range that covers and extends beyond the standard peak molecular weight range for the quantification of polytrehalose.

表6 -兩個經修改聚海藻糖之分子量分佈特徵 實施例17：藉由乾燥製備經高度純化聚海藻糖組成物The results in Table 6 below contain abbreviations for certain characteristics of molecular weight distribution. Gel permeation chromatography is represented by GPC, peak molecular weight is represented by PMW, weight average molecular weight is represented by WAMW, number average molecular weight is represented by NAMW, percentage distribution is represented by% distribution and molecular weight is represented by MW.

Table 6-Molecular weight distribution characteristics of two modified polytrehalose Example 17: Preparation of highly purified polytrehalose composition by drying

Approximately 100 mg of polytrehalose 1 was placed in the crucible. The crucible containing polytrehalose 1 was placed in an oven at 105°C for 30 minutes to produce a further purified polytrehalose composition, hereinafter referred to as polytrehalose 1'. The crucible containing the further purified polytrehalose 1'composition was removed from the oven and placed in a desiccator. Analysis of the further purified polytrehalose 1'composition in a humidity free atmosphere: analysis of total fucose and galactose by HPAE-PAD and analysis of total sulfur and relative ion content by ICP-MS and found to contain more than 99.9 % w/w total fucose, galactose, sulfate and relative ion content, in other words less than 0.1% impurities. Example 18 : Preparation of highly purified polytrehalose composition by drying

Place approximately 600 mg of polytrehalose 1 on the aluminum pan in the Ohaus MB 90 humidity analyzer instrument. The instrument was programmed to heat polytrehalose 1 at 105°C for 30 minutes to produce a further purified polytrehalose 1" composition. The further purified polytrehalose 1" composition was removed from the instrument and placed in a desiccator. Analyze samples in a free atmosphere of humidity: HPAE-PAD analysis of total fucose and galactose and ICP-MS analysis of total sulfur and relative ion content, and found to contain more than 99.9% w/w total fucose, The content of galactose, sulfate and relative ions is less than 0.1% impurities. Example 19 : Treatment of uterine horn fibrous adhesions with polytrehalose 1

To determine the efficacy of purified/modified polytrehalose 1 in inhibiting surgical adhesions, the following double uterine horn (DUH) surgery was performed on the two horns of a total of two New Zealand white rabbits. Before surgery, the rabbits were weighed and then prepared for surgery by preoperative medications of ketamine and xylazine.

Fucoidan solution was prepared at 0.33 mg/mL in Lactated Ringer's Injection USP (LRS) and sterilized by filtration. All instruments are sterile and kept sterile on site throughout the operation. Clean the abdomen and cut through the mid-abdomen to enter. The uterine horn is positioned, removed from the abdomen and scraped to induce injury. Also scrape close to the abdominal wall after scraping the uterine horn. Apply the lowest amount of fucoidan solution directly to the injured uterine horn and lateral wall area. Place the damaged uterine horn and abdominal wall close to each other and stabilize with sutures. 15 mL/kg fucoidan solution/rabbit weight was applied to the abdominal cavity, and then the incision was closed. The adhesion was evaluated two weeks after the operation. Measure the length of the uterine horn adhesion with a ruler. The uterine horn adhesion coverage percentage (the adhesion length as a percentage of the total damaged uterine horn length) is calculated as: Equation 2: Adhesion coverage (%) = 100 × uterine horn adhesion length f ÷ total injury uterine horn length

The same surgical method was applied to New Zealand white rabbits, receiving 15 mL/kg lactated Ringer's injection of USP (LRS) instead of fucoidan solution as a control group. Use Equation 2 to determine that the control group receiving LRS has 63% adhesion coverage. Table 7 shows the results obtained using the method described above for polytrehalose 2, which is a representative example of purified/modified polytrehalose. The results in the table below show the reduction in adhesion coverage relative to the control group.

表 7 ：使用聚海藻糖1之家兔子宮角粘連之減小Table 7 provides the results of treatment of six uterine horns with polytrehalose 1.

Table 7 : Reduction of uterine horn adhesion in rabbits using Polytrehalose 1

As can be seen from the results in Table 7, the purified/modified polytrehalose discussed herein can be used to successfully treat uterine horn adhesions after surgery. Example 20 : Treatment of uterine horn fibrous adhesions with polytrehalose 4

To determine the efficacy of purified/modified polytrehalose 4 in inhibiting surgical adhesions, the following double uterine horn (DUH) surgery was performed on two horns of a total of four New Zealand white rabbits. Prior to surgery, the rabbits were weighed and then prepared for surgery by pre-treatment with ketamine and xylazine.

Fucoidan solution was prepared at 3.75 mg/mL in Lactated Ringer's Injection USP (LRS) and sterilized by filtration. All instruments are sterile and kept sterile on site throughout the operation. Clean the abdomen and cut through the mid-abdomen to enter. The uterine horn is positioned, removed from the abdomen and scraped to induce injury. Also scrape close to the abdominal wall after scraping the uterine horn. 4 mL of fucoidan solution was directly applied to the injured uterine horn and lateral wall area on the left side and 4 mL of fucoidan solution was directly applied to the injured uterine horn and lateral wall area on the right side. Place the damaged uterine horn and abdominal wall close to each other and stabilize with sutures. The drainage tube is placed in the abdominal cavity, and then the incision is closed. The drainage tube was removed 48 hours after the operation. The adhesion was evaluated two weeks after the operation. Measure the length of the uterine horn adhesion with a ruler. Use Equation 2 to calculate uterine horn adhesion coverage.

The same surgical method was applied to three New Zealand white rabbits, receiving 4 mL/side of lactated Ringer's injection USP (LRS) instead of fucoidan solution as a control group. Use Equation 2 to determine that the control group receiving LRS has 73% adhesion coverage. Table 8 shows the results obtained using the method described above for polytrehalose 4, which is a representative example of purified/modified polytrehalose. The results in the table below show the reduction in adhesion coverage relative to the control group.

表 8 ：使用聚海藻糖4之家兔子宮角粘連之減小Table 8 provides the results of treating eight uterine horns with polytrehalose 4.

Table 8 : Reduction of uterine horn adhesion in rabbits using Polytrehalose 4

As can be seen from the results in Table 8, purified/modified polytrehalose can be used to successfully treat uterine horn adhesions after surgery. Example 21 : Treatment of uterine horn fibrous adhesions with polytrehalose 6

To determine the efficacy of purified/modified polytrehalose 6 in inhibiting surgical adhesions, the following double uterine horn (DUH) surgery was performed on two horns of a total of four New Zealand white rabbits. Prior to surgery, the rabbits were weighed and then prepared for surgery by pre-treatment with ketamine and xylazine.

Fucoidan solution was prepared at 0.33 mg/mL in Lactated Ringer's Injection USP (LRS) and sterilized by filtration. All instruments are sterile and kept sterile on site throughout the operation. Clean the abdomen and cut through the mid-abdomen to enter. The uterine horn is positioned, removed from the abdomen and scraped to induce injury. Also scrape close to the abdominal wall after scraping the uterine horn. Place the damaged uterine horn and abdominal wall close to each other and stabilize with sutures. Approximately 15 mL/kg of fucoidan solution/rabbit weight was applied to the abdominal cavity, and then the incision was closed. The adhesion was evaluated two weeks after the operation. Three rabbits were evaluated at each fucoidan concentration prepared. Measure the length of the uterine horn adhesion with a ruler. Use Equation 2 to calculate the uterine horn adhesion length.

The same surgical method was applied to 4 New Zealand white rabbits and received approximately 15 mL/kg control lactated Ringer's injection USP (LRS) instead of fucoidan solution. Equation 2 was used to determine that the control group receiving LRS had 71% adhesion coverage. Table 9 shows the results obtained for the polytrehalose 6 using the method described above. The results in the table below show the reduction in adhesion coverage relative to the control group.

表 9 ： 使用聚海藻糖9之家兔子宮角粘連相對於對照組LRS的增大Table 9 provides the results of treating eight uterine horns with polytrehalose 6.

Table 9 : Increase of LRS in rabbit uterine horn adhesion using Polytrehalose 9 compared with control group

As can be seen from the results in Table 9, polytrehalose 6 prepared using known methods and having a total non-polytrehalose content of polytrehalose greater than 50% w/w is not effective in treating fibrous adhesions. Example 22 : Treatment of uterine horn fibrous adhesions with polytrehalose 7

To determine the efficacy of purified/modified polytrehalose 7 in inhibiting surgical adhesions, the following double uterine horn (DUH) surgery was performed on two horns of a total of four New Zealand white rabbits. Prior to surgery, the rabbits were weighed and then prepared for surgery by pre-treatment with ketamine and xylazine.

Fucoidan solution was prepared at 0.33 mg/mL in Lactated Ringer's Injection USP (LRS) and sterilized by filtration. All instruments are sterile and kept sterile on site throughout the operation. Clean the abdomen and cut through the mid-abdomen to enter. The uterine horn is positioned, removed from the abdomen and scraped to induce injury. Also scrape close to the abdominal wall after scraping the uterine horn. Place the damaged uterine horn and abdominal wall close to each other and stabilize with sutures. Approximately 15 mL/kg of fucoidan solution/rabbit weight was applied to the abdominal cavity, and then the incision was closed. The adhesion was evaluated two weeks after the operation. Three rabbits were evaluated at each fucoidan concentration prepared. Measure the length of the uterine horn adhesion with a ruler. Use Equation 2 to calculate the uterine horn adhesion length.

The same surgical method was applied to 4 New Zealand white rabbits and received approximately 15 mL/kg control lactated Ringer's injection USP (LRS) instead of fucoidan solution. Use Equation 2 to determine that the control group receiving LRS has 76% adhesion coverage. Table 10 shows the results obtained for the polytrehalose 7 using the method described above. The results in the table below show the reduction in adhesion coverage relative to the control group.

表 10 ：使用聚海藻糖7之家兔子宮角粘連相對於對照組LRS的減小Table 10 provides the results of treating eight uterine horns with polytrehalose 7.

Table 10 : Reduced LRS of rabbit uterine horn adhesion using Polytrehalose 7 relative to the control group

As can be seen from the results in Table 10, polytrehalose 7 prepared using known methods and having a total non-polytrehalose content of polytrehalose greater than 50% w/w is not effective in treating fibrous adhesions. Example 23 : Treatment of uterine horn fibrous adhesions with polytrehalose 4

To determine the efficacy of purified/modified polytrehalose 4 in inhibiting surgical adhesions, the following double uterine horn (DUH) surgery was performed on two horns of a total of three New Zealand white rabbits. Prior to surgery, the rabbits were weighed and then prepared for surgery by pre-treatment with ketamine and xylazine.

Fucoidan solution was prepared at 5 mg/mL in Lactated Ringer's Injection USP (LRS) and sterilized by filtration. All instruments are sterile and kept sterile on site throughout the operation. Clean the abdomen and cut through the mid-abdomen to enter. The uterine horn is positioned, removed from the abdomen and scraped to induce injury. Also scrape close to the abdominal wall after scraping the uterine horn. Place the damaged uterine horn and abdominal wall close to each other and stabilize with sutures. The upper third and lower third of the muscle incision were closed and 5 mL/kg fucoidan solution/rabbit weight was applied to the abdominal cavity. The muscle incision was temporarily closed and the fucoidan solution was left in the abdominal cavity for 30 minutes. The muscle incision was opened again and the abdominal cavity was flushed with 10 mL/kg LRS. Aspirate most of the fluid in the abdominal cavity, and then close the incision. Adhesion formation was evaluated two weeks after surgery. Measure the length of the uterine horn adhesion with a ruler. The uterine horn adhesion coverage percentage (which is the length of the adhesion as a percentage of the total damaged uterine horn length) is calculated using Equation 2.

Table 11 shows the results obtained using the method described above for polytrehalose 4, which is a representative example of purified/modified polytrehalose. The results in the table below show the average adhesion length of the 6 uterine horns that were scored.

表 11 ：使用聚海藻糖4之粘連長度Table 11 provides the results of treatment of six uterine horns with polytrehalose 4.

Table 11 : Adhesion length using polytrehalose 4

As can be seen from the results in Table 11, purified/modified polytrehalose can be used to successfully inhibit, prevent, remove, reduce, or otherwise treat postoperative uterine horn adhesions. Example 24 : Treatment of uterine horn fibrous adhesions with purified / modified polytrehalose composition

To determine the efficacy of the purified/modified polytrehalose composition containing 92% w/w total fucose, galactose, sulfate and relative ions in inhibiting surgical adhesion, a total of twenty New Zealand white rabbits The following two uterine horn (DUH) procedures were performed on both horns. Before the operation, the rabbit was weighed and then prepared for the surgery by preoperative medications of midazolam and dexmeditomidine.

Fucoidan solutions were prepared in lactated Ringer's injection USP (LRS) at various concentrations of 0.02 mg/mL, 0.1 mg/mL, 0.5 mg/mL or 2.5 mg/mL, and sterilized by filtration. All instruments are sterile and kept sterile on site throughout the operation. Clean the abdomen and cut through the mid-abdomen to enter. The uterine horn is positioned, removed from the abdomen and scraped to induce injury. Also scrape close to the abdominal wall after scraping the uterine horn. Place the damaged uterine horn and abdominal wall close to each other and stabilize with sutures. Approximately 2 mL/kg fucoidan solution/rabbit weight was applied to the abdominal cavity, and then the incision was closed. The adhesion was evaluated two weeks after the operation. Five rabbits were treated and evaluated for the concentration of each fucoidan prepared. Measure the length of the uterine horn adhesion with a ruler. Use Equation 2 to calculate the uterine horn adhesion length.

表 12 ： 使用經純化/經修改聚海藻糖組成物之家兔子宮角粘連相對於對照LRS的減小The same surgical method was applied to 5 additional New Zealand white rabbits for control, each receiving approximately 2 mL/kg of control lactated Ringer's injection USP (LRS) instead of the fucoidan solution. Use Equation 2 to determine that the control group receiving LRS has 100% adhesion coverage. Table 12 shows the results obtained using the method described above for the purified/modified polytrehalose composition at different concentrations and doses (a total of forty uterine horns were treated, purified/modified poly at each concentration Ten trehalose compositions each); the results are shown as a reduction in adhesion coverage relative to the control group.

Table 12 : Decrease of uterine horn adhesion in rabbits using purified/modified polytrehalose composition relative to control LRS

As can be seen from the results in Table 12, purified/modified polytrehalose can be used to successfully inhibit, prevent, remove, reduce, or otherwise treat postoperative uterine horn adhesions.

Unless the context or definition clearly indicates otherwise, all terms used herein are used according to their general meaning. Likewise, unless expressly indicated otherwise, the use of "or" in the present invention includes "and" and vice versa. Unless expressly stated otherwise or clearly indicated by context, non-limiting terms should not be construed as limiting (eg, "including", "having" and "including" typically indicate "including (but not limited to)"). Unless clearly stated otherwise or clearly indicated by the context, singular forms such as "a (an)" and "the" (including in the scope of patent application) include a plurality of things referred to.

Unless otherwise stated, adjectives (such as "substantially" and "approximately") that modify the conditions or relationship characteristics of one or more features of an embodiment indicate that the condition or characteristic is limited to the embodiment for which it is intended to apply Within the acceptable tolerance of the operation.

The scope of the method, composition, system, etc. of the present invention includes both method plus function and step plus function concept. However, unless the term "method" is specifically mentioned in the scope of patent application, the scope of patent application should not be interpreted as indicating the "method plus function" relationship, and should be included in the term "method" in the scope of patent application. The specific description is interpreted in time to indicate the "method plus function" relationship. Similarly, unless the term "step" is specifically mentioned in the scope of patent application, the scope of patent application should not be interpreted as indicating the "step plus function" relationship, and should be included in the term "step" in the scope of patent application. The specific description is timely interpreted as a "step plus function" relationship.

It will be understood from the foregoing that, although specific embodiments have been discussed herein for illustrative purposes, various modifications may be made without departing from the spirit and scope of the discussions herein. Therefore, the system and method of this case include such modifications and all permutations and combinations of the subject matter described herein and are not limited, except for those that are fully supported by the scope of the attached patent application or in the discussion and drawings herein Beyond those who advocate restrictions.

1200: Cation content modification system 1202: Input supply pipeline 1204: Pre-filter 1206: Cation content modification system output valve 1208: Cation content modification system output pipeline 1210: TFF filter 1212: TFF supply pipeline 1214: TFF input pump 1216: Polytrehalose container 1217: TFF Retentate Valve 1218: TFF retentate return pipeline 1219: TFF filtered output line 1220: The first diafiltration solution container 1224: First diafiltration solution valve 1225: The first diafiltration solution supply line 1230: Second diafiltration solution container 1234: Second diafiltration solution valve 1235: Second diafiltration solution supply line

FIG. 1 schematically depicts an exemplary system for modifying the cation content of a starting polytrehalose composition and using a chelating agent to remove low molecular weight non-polytrehalose components in tangential flow filtration. Figure 2A depicts NMR results, which show that certain polytrehaloses treated according to the methods herein undergo structural changes in polytrehalose. Figure 2B depicts 2-D NMR results, which demonstrate that certain polytrehalose treated according to the methods herein undergo structural changes in polytrehalose. The drawings present exemplary embodiments of aspects of the compositions, methods, etc. of the present invention. The implementation of the systems, methods, etc. herein may include other features or steps not shown in the drawings. In addition, the examples set forth herein illustrate embodiments of such systems, methods, etc. in one or more forms and such examples are not to be considered as limiting the scope of the invention in any way. The embodiments herein are not exhaustive and do not limit the disclosure herein to the precise forms disclosed, such as the detailed description below.

1200: Cation content modification system

1202: Input supply pipeline

1204: Pre-filter

1206: Cation content modification system output valve

1208: Cation content modification system output pipeline

1210: TFF filter

1212: TFF supply pipeline

1214: TFF input pump

1216: Polytrehalose container

1217: TFF Retentate Valve

1218: TFF retentate return pipeline

1219: TFF filtered output line

1220: The first diafiltration solution container

1224: First diafiltration solution valve

1225: The first diafiltration solution supply line

1230: Second diafiltration solution container

1234: Second diafiltration solution valve

1235: Second diafiltration solution supply line

Claims (65)

A polytrehalose composition consisting of at least about 96% w/w polytrehalose and no more than about 4% non-polytrehalose components or compounds or substances, wherein the polytrehalose must have fucose and sulfate And relative ions, and can be further composed of galactose, glucose, rhamnose, mannose, xylose, and glucuronic acid. The polytrehalose composition according to claim 1, wherein the polytrehalose contains not more than about 17% relative ions, and wherein the total content of fucose, galactose, sulfate and relative ions in the polytrehalose Greater than 96% w/w. The polytrehalose composition according to claim 1, wherein the polytrehalose consists of the following: fucose, galactose, sulfate, and relative ions. The polytrehalose composition according to claim 1, wherein the impurities include water. The poly-trehalose composition according to any one of claims 1 to 4, wherein the poly-trehalose composition is composed of the poly-trehalose and not more than about 3% of such impurities. The poly-trehalose composition according to any one of claims 1 to 4, wherein the poly-trehalose composition is composed of the poly-trehalose and not more than about 1% of such impurities. The poly-trehalose composition according to any one of claims 1 to 4, wherein the poly-trehalose composition is composed of the poly-trehalose and such impurities not exceeding about 0.1%. The polytrehalose composition according to any one of claims 1 to 4, wherein the fucose content in the polytrehalose is greater than 25% w/w. The polytrehalose composition according to any one of claims 1 to 4, wherein the fucose content in the polytrehalose is greater than 35% w/w. The polytrehalose composition according to any one of claims 1 to 4, wherein the content of galactose in the polytrehalose is greater than 10% w/w. The polytrehalose composition according to any one of claims 1 to 4, wherein the total relative ion content in the polytrehalose is between 7% w/w and 14% w/w. The polytrehalose composition according to any one of claims 1 to 4, wherein the relative ion consists essentially of at least one of sodium and potassium. The polytrehalose composition according to any one of claims 1 to 4, wherein the polytrehalose has a molecular weight distribution, wherein when measured using an aqueous gel permeation chromatography setting, at least 60% w/w If the distribution is greater than 100 kDa, the aqueous gel permeation chromatography setup basically consists of the following: A 300 mm analytical gel permeation chromatography column with an inner diameter of 7.8 mm, filled with a hydroxylated polymethacrylate-based gel, having an effective molecular weight range between about 50 kDa and about 5,000 kDa; one 300 mm analytical gel permeation chromatography column with an inner diameter of 7.8 mm, filled with hydroxylated polymethacrylate-based gel, with an effective molecular weight range between about 1 kDa and about 6,000 kDa; and one A 40 mm protective column with an inner diameter of 6 mm, filled with a gel based on hydroxylated polymethacrylate, the two analytical gel permeation chromatography columns and the one protective column are contained at about 30°C In the lower column chamber; Refractive index detector at about 30℃; 0.1M sodium nitrate mobile phase at 0.6 mL/min; and Relative to the peak molecular weight standard curve, the peak molecular weight standard curve basically consists of the following: the first polydextrose standard with a peak molecular weight of about 2,200 kDa; the second polydextrose standard with a peak molecular weight between about 720 kDa and about 760 kDa Between; the third polydextrose standard, the peak molecular weight is between about 470 kDa and about 510 kDa; the fourth polydextrose standard, the peak molecular weight is between about 370 kDa and about 410 kDa; the fifth polydextrose standard, the peak molecular weight is about 180 kDa and about 220 kDa; and the sixth polydextrose standard, the peak molecular weight is between about 40 kDa and 55 kDa. The polytrehalose composition according to claim 13, wherein the polytrehalose has a molecular weight distribution, wherein at least 92% w/w of the distribution is greater than 100 kDa. The poly-trehalose composition according to claim 13, wherein the poly-trehalose has a molecular weight distribution, wherein at least 97% w/w of the distribution is greater than 100 kDa. The polytrehalose composition according to claim 13, wherein the polytrehalose has a weight average molecular weight greater than 100 kDa. The polytrehalose composition of any one of claims 1 to 4, wherein the polytrehalose has a sulfation level between about 35% w/w and 60% w/w. The poly-trehalose composition according to any one of claims 1 to 4, wherein the poly-trehalose has a total carbohydrate content of between 27% w/w and 80% w/w, and wherein the poly-trehalose The total sugar glucuronic acid, mannose, rhamnose, glucose and xylose content of the total carbohydrate content is less than about 12% w/w. The polytrehalose composition according to any one of claims 1 to 4, wherein when the polytrehalose composition is dissolved in water at a concentration of 50 mg/mL, it has a temperature between about 4 cP and 50 cP Viscosity. The polytrehalose composition according to any one of claims 1 to 4, wherein the polytrehalose contains less than 5% w/w acetyl content. The polytrehalose composition according to any one of claims 1 to 4, wherein the polytrehalose contains less than 2% w/w acetyl content. The polytrehalose composition according to any one of claims 1 to 4, wherein when 2D 1H-13C heteronuclear multiple quantum coherence (heteronuclear multiple quantum coherence) at 70 ℃ with solvent signal suppression is equipped On a 600 MHz spectrometer with a 5mm cold probe, in a carbon size range of 10-30 ppm, with 8 increments of 256-512 scans per measurement, the polytrehalose contains substantially 0% w/w The acetyl content. A method comprising preparing the polytrehalose composition according to any one of claims 1 to 4. A method comprising using the polytrehalose composition according to any one of claims 1 to 4. Use of the polytrehalose composition according to any one of claims 1 to 4 for the manufacture of pharmaceutical products for the treatment of fibrous adhesions. A medically acceptable composition comprising a therapeutically effective amount of the polytrehalose composition according to any one of claims 1 to 4 in a medically acceptable buffer or diluent. A use of a medically acceptable composition as described in claim 26 for the manufacture of a medicinal product for the treatment of a disease or disease in an animal, wherein the medicinal product is used for administration to the animal A therapeutically effective amount of the polytrehalose between 0.5 mg/kg and 50 mg/kg. The use according to claim 27, wherein the amount is between about 1 mg/kg and 5 mg/kg. The use according to claim 27, wherein the amount is between about 1.5 mg/kg and 3 mg/kg. The use as claimed in claim 27, wherein the amount is between about 5 mg/kg and 10 mg/kg. The use as claimed in claim 27, wherein the condition or disease is fibrous adhesion at the target site of the animal, and wherein the administration comprises administering the therapeutically effective amount to the target site. A use of a medically acceptable composition as described in claim 26 for the manufacture of a medicinal product for the treatment of an animal's pathology or disease, wherein the medicinal product is used for administration to the animal at about 0.04 A therapeutically effective amount of the polytrehalose between mg/kg and 25 mg/kg. The use according to claim 32, wherein the amount is between about 0.2 mg/kg and 10 mg/kg. A medical composition comprising the polytrehalose composition according to any one of claims 1 to 4 between about 0.02 mg/mL and 100 mg/mL, wherein the medical device is configured or constructed to treat Animal diseases or conditions. The medical composition of claim 34, comprising the polytrehalose composition between about 0.5 mg/mL and 5 mg/mL. The medical composition of claim 34, which contains about 2.5 mg/mL of the polytrehalose composition. The medical composition according to claim 34, wherein the medical composition is a medical device. The medical composition according to claim 34, wherein the disease or condition is fibrous adhesion. Use of the medical composition according to any one of claims 34 to 38 for the manufacture of a medicinal product for the treatment of a selected disease or condition in a patient, wherein the medicinal product is used to contain the following In the method: Identify the selected target site in the patient's body (including or fairly susceptible to the selected disease or condition) and then administer the drug to the target site in the patient's body, where the target site is the surgical site and is below Perform at least one of the administrations to treat the selected disease or condition: after opening the surgical wound at the surgical site, b) during the surgery and c) after closing the surgical wound. The use according to claim 39, wherein the disease or condition is fibrous adhesion. The use according to claim 39, wherein the administration is performed after surgery and before closing the surgical wound. The use according to claim 39, wherein the investment takes less than 2 minutes. A method for removing impurities from a starting polytrehalose composition to obtain a purified/modified polytrehalose composition, comprising: Provide the starting polytrehalose composition containing impurities; Adding a flocculation aid to the starting polytrehalose composition to produce a reaction mixture; Flocculate the impurities by heating the reaction mixture to produce flocculated impurities; and Remove such flocculated impurities. The method according to claim 43, wherein the flocculation aid contains a salt. The method according to claim 43, wherein the flocculation aid comprises alkali. A method for removing impurities from a starting polytrehalose composition to obtain a purified/modified polytrehalose composition, comprising: Provide a solid starting polytrehalose composition and an extraction medium that cannot dissolve polytrehalose (configured to dissolve impurities); Mixing the starting polytrehalose composition with the extraction medium to produce a mixture of the purified/modified polytrehalose and the extraction medium; and The purified/modified polytrehalose composition is separated from the extraction medium. The method of claim 46, wherein the extraction medium comprises at least one organic solvent having a relative polarity of less than 0.765. The method of claim 47, wherein the extraction medium further comprises at least one of the following: alkali, detergent, and oxidant. A method for removing impurities from a starting polytrehalose composition to obtain a purified/modified polytrehalose composition, comprising: The starting polytrehalose composition containing impurities contained in the solution, including suspended impurities; Using an ionic polyvalent impurity precipitant to precipitate these impurities from the solution, thereby producing a mixture of suspended impurities, precipitated impurities and supernatant solution; and The suspended impurities and Shen impurities are separated from the supernatant solution. The method according to claim 49, wherein the ionic polyvalent impurity precipitating agent contains a salt of a divalent or trivalent cation. The method according to claim 49, wherein the ionic polyvalent impurity precipitating agent contains a base of a divalent or trivalent cation. The method according to any one of claims 49 to 51, wherein separating the suspended impurities and the precipitated impurities from the supernatant solution includes by adding to the mixture of suspended impurities, precipitated impurities and the supernatant solution Flocculant to flocculate these suspended impurities and sediment impurities. A method for removing impurities from a starting polytrehalose composition to obtain a purified/modified polytrehalose composition, comprising: Provide the starting polytrehalose composition containing impurities; Adjust the pH of the starting polytrehalose composition to between about 8 and 14; Adding a cell disrupting agent configured for lysing cell components to the starting polytrehalose to produce a reaction mixture containing the cell disrupting agent, biomolecule lysate and the starting polytrehalose composition; and The cell disrupting agent and biomolecule lysate are removed from the reaction mixture. The method of claim 53, wherein the cell disrupting agent comprises a cleaning agent. The method of claim 53 or 54, wherein removing the cell disrupting agent and biomolecule lysate comprises adding a flocculant to the reaction mixture, the flocculating agent configured to dissolve the cell disrupting agent and biomolecule Cell products flocculate. The method of claim 53 or 54, wherein removing the cell disrupting agent comprises adding a precipitating agent to the reaction mixture, the precipitating agent configured to render the cell disrupting agent insoluble in the reaction mixture, thereby producing Precipitate. The method of claim 53 or 54, wherein removing the biomolecule lysate includes adding a precipitant to the reaction mixture, the precipitant configured to render the biomolecule lysate insoluble in the In the reaction mixture, a precipitate is produced. The method according to claim 54, wherein removing the cleaning agent comprises: Dilute the reaction mixture until the concentration of the cleaning agent is lower than the predetermined concentration; and The reaction mixture containing the cleaning agent is subjected to diafiltration through a tangential flow filter filter having a molecular weight cutoff higher than the maximum molecular weight of the cleaning agent. A method for removing impurities from a starting polytrehalose composition to obtain a purified/modified polytrehalose composition, comprising: The starting polytrehalose composition containing impurities provided in the aqueous starting solution; Mixing the aqueous starting solution with an organic solvent to produce an aqueous-organic phase mixture; and The aqueous phase-organic phase mixture is separated to obtain an aqueous portion and an organic portion. The method according to claim 59, wherein the organic solvent comprises at least one organic solvent having a relative polarity of less than 0.765. A method for modifying the cation content of the starting polytrehalose composition, the method comprising: The starting polytrehalose composition provided in the starting solution; and The starting solution was diafiltered through the tangential flow filter filter using a solution of the chelating agent across the tangential flow filter filter to produce a retentate polytrehalose composition. The method of claim 61, wherein the retained polytrehalose composition comprises a cation content consisting essentially of sodium and/or potassium. A method for removing impurities from a starting polytrehalose composition to obtain a purified/modified polytrehalose composition, comprising: Provide the starting polytrehalose composition containing impurities; Subjecting the starting polytrehalose composition to a suitable pressure above 70 bar and a suitable temperature above 30°C in a supercritical extractor; and Filling the supercritical extractor with supercritical fluid to remove impurities into the supercritical fluid; and After a predetermined amount of time, the supercritical fluid containing the extracted impurities is removed. The method of claim 63, wherein the pressure is between about 70 bar and about 2000 bar. The method of claim 63, wherein the temperature is between about 30°C and about 300°C.

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